Supplementary Fig. 1
Myricetin attenuates endoplasmic reticulum (ER) stress induced by sarcoendoplasmic reticulum calcium ATPase (SERCA) inhibition and apoptosis in INS-1 cells. (A–D) INS-1 cells were incubated with 0.5 µM thapsigargin (TG) in the presence or absence of the indicated concentrations of myricetin for 24 hours. (A) Representative images of Western blot analysis of ER stress markers: glucose regulated protein 78 (Grp78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (P-PERK), phosphorylated eukaryotic initiation factor 2α (P-eIF2α), activating transcription factor 4 (ATF4), and CCAAT-enhancer-binding protein homologous protein (CHOP). (B) Representative flow cytometry analysis images of mitochondrial membrane potential observed with 3,3′-dihexyloxacarbocyanine iodide (DiOC6) dye. (C) Representative images of Western blot analysis of cytochrome c in the cytosol, cleaved caspase-3 (C-Capase 3), and B-cell lymphoma 2 (Bcl-2). (D) Cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. All data are expressed as the mean±standard deviation of at least three independent experiments. aP<0.05 vs. control, bP<0.001 vs. control, cP<0.01 vs. TG, dP<0.001 vs. TG, eP<0.01 vs. control, fP<0.05 vs. TG, gP<0.005 vs. TG.
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Fig. 1Myricetin protects INS-1 cells and isolated rat islets from high glucose (HG)-induced apoptosis. (A) Chemical structure of myricetin: carbon numbering is indicated. (B) INS-1 cells were treated with the indicated concentrations of myricetin for 24 hours. Cell viability was measured using the Cell Counting Kit-8 (Dojindo Laboratories). (C, D) INS-1 cells (C) and isolated rat islets (D) were incubated with 30 mM glucose (HG) in the presence or absence of the indicated concentrations of myricetin for 24 hours (C) or 48 hours (D), respectively. Cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. All data are expressed as the mean±standard deviation of at least three independent experiments. aP<0.001 vs. control, bP<0.005 vs. HG, cP<0.05 vs. HG.
Fig. 2Myricetin attenuates mitochondrial dysfunction in INS-1 cells exposed to high glucose (HG). (A–D) INS-1 cells were incubated with 30 mM glucose (HG) in the presence or absence of myricetin for 24 hours. (A) Intracellular reactive oxygen species (ROS) production was measured using 2′, 7′-dichlorodihydrofluorescein diacetate (DCF-DA). Data are expressed as the mean±standard deviation of at least three independent experiments. (B) Representative flow cytometry analysis images of the mitochondrial membrane potential observed with the 3,3′-dihexyloxacarbocyanine iodide (DiOC6) dye. (C) Representative image of Western blot analysis of cytochrome c in cytosol and cleaved caspase-3 (C-caspase 3). (D) Representative images of Western blot analysis of Bax/B-cell lymphoma 2 (Bcl-2). aP<0.01 vs. control, bP<0.05 vs. HG, cP<0.05 vs. control, dP<0.001 vs. control, eP<0.001 vs. HG.
Fig. 3Myricetin inhibits cyclin-dependent kinase 5 (CDK5) in high glucose (HG)-exposed INS-1 cells. (A) INS-1 cells were incubated with 30 mM glucose (HG) in the presence or absence of roscovitine for 24 hours. Cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Data are expressed as the mean±standard deviation of at least three independent experiments. (B) INS-1 cells were incubated with 30 mM glucose (HG) with myricetin or roscovitine for 24 hours. Representative images of western blot analysis of cleaved caspase-3. (C) INS-1 cells were incubated with 30 mM glucose with or without myricetin for different time periods. Representative images of Western blot analysis of CDK5 phosphorylated at tyrosine 15 (Tyr15) and p35. (D) The proposed binding model of myricetin to CDK5 based on docking studies. The dotted lines indicate hydrogen bonds interactions. aP<0.01 vs. control, bP<0.05 vs. HG, cP<0.001 vs. control, dP<0.005 vs. HG, eP<0.001 vs. HG, fP<0.05 vs. control.
Fig. 4Myricetin counteracts the decrease in total and nuclear levels of pancreatic duodenal homeobox 1 (PDX1) in high glucose (HG)-exposed INS-1 cells. (A, B) INS-1 cells were incubated with 30 mM glucose (HG) in the presence or absence of myricetin for 24 hours. (A) PDX1 mRNA levels were determined by real-time polymerase chain reaction (PCR). Data are expressed as the mean±standard deviation of at least three independent experiments. (B) Representative images of Western blot analysis of PDX1. (C) INS-1 cells were incubated with 30 mM glucose (HG) in the presence or absence of roscovitine for 24 hours. Representative images of western blot analysis of PDX1. (D) INS-1 cells were incubated with 30 mM glucose (HG) along with myricetin for 24 hours. Representative images of the subcellular localization of PDX1 by confocal microscope. 4′,6-Ddiamidino-2-phenylindole (DAPI) was used to stain the nuclei. aP<0.05 vs. control, bP<0.001 vs. control, cP<0.05 vs. HG, dP<0.01 vs. HG, eP<0.01 vs. control.
Fig. 5Myricetin conterbalances the decrease in sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) expression and prevents endoplasmic reticulum (ER) stress in INS-1 cells exposed to high glucose (HG). (A, B) INS-1 cells were incubated with 30 mM glucose (HG) in the presence or absence of myricetin for 24 hours. (A) SERCA2b mRNA levels were determined by real-time polymerase chain reaction (PCR). Data are expressed as the mean±standard deviation of at least three independent experiments. (B) INS-1 cells were incubated with 30 mM glucose (HG) with myricetin for 24 hours. Representative images of Western blot analysis of SERCA2b. (C, D) INS-1 cells were incubated with 30 mM glucose (HG) in the presence or absence of myricetin for 24 hours. Representative images of Western blot analysis of (C) ER stress markers: glucose regulated protein 78 (Grp78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (P-PERK), phosphorylated eukaryotic initiation factor 2α (P-eIF2α), activating transcription factor 4 (ATF4), and CCAAT-enhancer-binding protein homologous protein (CHOP). (D) Phosphorylated c-Jun N-terminal kinase (P-JNK). aP<0.05 vs. control, bP<0.05 vs. HG, cP<0.01 vs. control, dP<0.005 vs. control, eP<0.005 vs. HG, fP<0.01 vs. HG, gP<0.001 vs. control, hP<0.001 vs. HG.
Fig. 6Myricetin effect on insulin mRNA and glucose-stimulated insulin secretion (GSIS). (A) INS-1 cells were incubated with 30 mM glucose (high glucose [HG]) in the presence or absence of myricetin for 24 hours and insulin mRNA levels was determined by real-time polymerase chain reaction (PCR). Data are expressed as the mean±standard deviation of at least three independent experiments. (B) GSIS was measured by rat insulin radioimmunoassay as described in the methods section. aP<0.001 vs. control, bP<0.05 vs. control, cP<0.05 vs. HG, dP<0.05 vs. 16.6 mM glucose control.