Fig. 1Pioglitazone (Pio) improves palmitate (PA)-induced β-cell impairment via repression of the inflammatory response and endoplasmic reticulum (ER) stress. (A) Mouse insulinoma 6 (MIN6) cells were incubated with 0.5 mM PA in the presence or absence of 10 µM Pio for 24 hours, and the glucose-stimulated (1 or 4.5 g/L) insulin secretion of MIN6 cells was evaluated. Secreted insulin was measured by a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit. The values are representative of 6 independent experiments. (B) Expression of cleaved caspase-3 (c-casp3), an apoptotic protein, was measured by Western blot analysis. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is represented as the percentage of relative absorbance compared to the vehicle group. (D) Transcription of tumor necrosis factor α (TNFA), interleukin 6 (IL6), and IL1B was measured by real time reverse-transcription polymerase chain reaction, and normalized with β-actin. (E) The ER stress proteins, including phosphor-eukaryotic translation initiation factor 2α (p-eIF2α), glucose-regulated protein 78 (GRP78), cleaved-activating transcription factor 6 (c-ATF6), and C/EBP homologous protein (CHOP), were measured by Western blot analysis and (F) the ratio of p-eIF2α, GRP78, and CHOP to β-actin was described. Each value represents the mean of three experiments. t-casp3, total caspase-3; Veh, vehicle; PA, palmitate. aP<0.01, bP<0.001 compared with the vehicle group; cP<0.05, dP<0.001 compared with the PA group.
Fig. 2Pioglitazone (Pio) represses lipopolysaccharide (LPS)-induced inflammatory response and endoplasmic reticulum (ER) stress. The mouse insulinoma 6 (MIN6) cells were incubated with 10 µg/mL LPS in the presence or absence of 10 µM Pio for 8 hours. (A) Phosphorylation of nuclear factor-kappa B (NF-κB) and jun N-terminal kinase (JNK), essential factors of the inflammatory response, was evaluated by Western blot. (B) The density of phosphorylated NF-κB and JNK was normalized to that of the total forms. (C) The inflammatory cytokines were measured by reverse-transcription polymerase chain reaction, and normalized with β-actin. (D) The ER stress proteins, including phosphor-eukaryotic translation initiation factor 2α (p-eIF2α), glucose-regulated protein 78 (GRP78), and C/EBP homologous protein (CHOP), were measured by Western blot analysis and (E) the ratio to β-actin was described. Each value represents the mean of three experiments. t-NF-κB, total nuclear factor kappa-light-chain-enhancer of activated B cells; p-JNK, phosphorylated c-Jun N-terminal kinase; t-JNK, total c-Jun N-terminal kinase; TNFα, tumor necrosis factor α; IL, interleukin. aP<0.05, bP<0.01, cP<0.001 compared with the vehicle (Veh) group; dP<0.05, eP<0.01, fP<0.001 compared with the LPS group.
Fig. 3Pioglitazone (Pio) reduces endoplasmic reticulum (ER) stress, but not the inflammatory response in tunicamycin (TU)-challenged mouse insulinoma 6 (MIN6) cells. The MIN6 cells were incubated with 2 µg/mL TU in the presence or absence of 10 µM Pio for 24 hours. (A) The ER stress proteins, including phosphor-eukaryotic translation initiation factor 2α (p-eIF2α), glucose-regulated protein 78 (GRP78), cleaved-activating transcription factor 6 (c-ATF6), and C/EBP homologous protein (CHOP), were measured by Western blot analysis, and (B) the ratio to β-actin was described. (C) The transcription of tumor necrosis factor α (TNFA), interleukin 6 (IL6), and IL1B was measured by real-time reverse-transcription polymerase chain reaction, and normalized with β-actin. Each value represents the mean of three experiments. aP<0.05, bP<0.01, cP<0.001 compared with the vehicle (Veh) group; dP<0.01, eP<0.001 compared with the TU group.
Fig. 4Pioglitazone (Pio) protects pancreatic β-cells and regulates blood glucose levels in high-fat (HF)-diet-induced diabetic mice. The pancreatic islet from KK-Ay mice, which were fed an HF diet with or without Pio, were examined by double-immunofluorescence for (A) insulin-activating transcription factor 6 and insulin-glucose-regulated protein 78 (GRP78), (B) insulin-monocyt e chemoattractant protein-1 and insulin-glucagon (GLU), (C) insulin-glucose transporter 2 (GLUT2). (A-C) The nucleus was visualized by 4′,6-diamidino-2-phenylindole (DAPI). Bars=20 µm. (D) Blood glucose levels and body weight were measured every week. INS, insulin; ATF6, activating transcription factor 6; MCP1, monocyte chemoattractant protein-1. aP<0.05 compared with the HF group.