Metformin induces suppression of NAD(P)H oxidase activity in podocytes.
Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms. The experiments were performed in normal (NG, 5.6mM) and high (HG, 30mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-(3)H]-glucose. ROS production increased by about 27% (187+/-8 vs. 238+/-9 arbitrary units AU, P<0.01) in HG. Metformin (2mM, 2h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58+/-0.07 to. 0.99+/-0.06, and from 0.53+/-0.03 to 0.64+/-0.03; P<0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C. We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK.
Piwkowska
Agnieszka
A
Mossakowski Medical Research Centre Polish Academy of Sciences, Gdańsk, Poland. apiwkowska@cmdik.pan.pl
Rogacka
Dorota
D
Jankowski
Maciej
M
Dominiczak
Marek Henryk
MH
Stepiński
Jan Kazimierz
JK
Angielski
Stefan
S
D000091162:AMP-Activated Protein Kinase Kinases; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002460:Cell Line; D005947:Glucose / Q000378:metabolism; D006943:Hyperglycemia / Q000201:enzymology*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008687:Metformin / Q000494:pharmacology*; D051379:Mice; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D050199:Podocytes / Q000187:drug effects* / Q000276:immunology; D011494:Protein Kinases / Q000378:metabolism; D013481:Superoxides / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000975:Antioxidants; D007004:Hypoglycemic Agents; D013481:Superoxides; D008687:Metformin; D019255:NADPH Oxidases; D011494:Protein Kinases; D000091162:AMP-Activated Protein Kinase Kinases; D005947:Glucose