title: Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells.
abstract: OBJECTIVE
  Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress.
  
  
  METHODS
  Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity.
  
  
  RESULTS
  High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects.
  
  
  CONCLUSIONS
  Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications.
authors.affiliation: Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: battsetseg.batchuluun@gmail.com.
authors.forename: Battsetseg
authors.initials: B
authors.lastname: Batchuluun
authors.affiliation: Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: toyoshi@intmed3.med.kyushu-u.ac.jp.
authors.forename: Toyoshi
authors.initials: T
authors.lastname: Inoguchi
authors.affiliation: Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: noriyuki@intmed3.med.kyushu-u.ac.jp.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sonoda
authors.affiliation: Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: ssasaki@intmed3.med.kyushu-u.ac.jp.
authors.forename: Shuji
authors.initials: S
authors.lastname: Sasaki
authors.affiliation: Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: tomo-i@intmed3.med.kyushu-u.ac.jp.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Inoue
authors.affiliation: Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: fujimu@redoxnavi.med.kyushu-u.ac.jp.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Fujimura
authors.affiliation: Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: daipon@agr.kyushu-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Miura
authors.affiliation: Department of Internal Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: takayana@intmed3.med.kyushu-u.ac.jp.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Takayanagi
mesh_terms: D000166:Acridines / Q000737:chemistry; D001011:Aorta / Q000166:cytology; D001665:Binding Sites; D002478:Cells, Cultured; D004075:Diglycerides / Q000737:chemistry; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D052216:Glucagon-Like Peptide 1 / Q000031:analogs & derivatives* / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry*; D000069450:Liraglutide; D049449:Luminescence; D008687:Metformin / Q000737:chemistry*; D019255:NADPH Oxidases / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D000166:Acridines; D004075:Diglycerides; D007004:Hypoglycemic Agents; D017382:Reactive Oxygen Species; C033472:10,10'-dimethyl-9,9'-biacridinium; D000069450:Liraglutide; D052216:Glucagon-Like Peptide 1; D008687:Metformin; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D005947:Glucose
keywords: AMPK; DAG; Endothelial cell; High glucose; Liraglutide; Metformin; Oxidative stress; PKC