Myricetin Protects Against High Glucose-Induced β-Cell Apoptosis by Attenuating Endoplasmic Reticulum Stress via Inactivation of Cyclin-Dependent Kinase 5

Abstract

Background: Chronic hyperglycemia has deleterious effects on pancreatic β-cell function and turnover. Recent studies support the view that cyclin-dependent kinase 5 (CDK5) plays a role in β-cell failure under hyperglycemic conditions. However, little is known about how CDK5 impair β-cell function. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. In this study, we examined the effect of myricetin on high glucose (HG)-induced β-cell apoptosis and explored the relationship between myricetin and CDK5.

Methods: To address this question, we subjected INS-1 cells and isolated rat islets to HG conditions (30 mM) in the presence or absence of myricetin. Docking studies were conducted to validate the interaction between myricetin and CDK5. Gene expression and protein levels of endoplasmic reticulum (ER) stress markers were measured by real-time reverse transcription polymerase chain reaction and Western blot analysis.

Results: Activation of CDK5 in response to HG coupled with the induction of ER stress via the down regulation of sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) gene expression and reduced the nuclear accumulation of pancreatic duodenal homeobox 1 (PDX1) leads to β-cell apoptosis. Docking study predicts that myricetin inhibit CDK5 activation by direct binding in the ATP-binding pocket. Myricetin counteracted the decrease in the levels of PDX1 and SERCA2b by HG. Moreover, myricetin attenuated HG-induced apoptosis in INS-1 cells and rat islets and reduce the mitochondrial dysfunction by decreasing reactive oxygen species production and mitochondrial membrane potential (ΔΨm) loss.

Conclusion: Myricetin protects the β-cells against HG-induced apoptosis by inhibiting ER stress, possibly through inactivation of CDK5 and consequent upregulation of PDX1 and SERCA2b.

Keywords: Apoptosis; Cyclin-dependent kinase 5; Endoplasmic reticulum stress; Insulin-secreting cells; Myricetin.

Fig. 1. Myricetin 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. 2. Myricetin 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. 3. Myricetin 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. 4. Myricetin 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. 5. Myricetin 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. 6. Myricetin 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.