Protection of INS-1 cells from free fatty acid-induced apoptosis by targeting hOGG1 to mitochondria.
Chronic exposure to elevated levels of free fatty acids (FFAs) impairs pancreatic beta-cell function and contributes to the decline of insulin secretion in type 2 diabetes. Previously, we reported that FFAs caused increased nitric oxide (NO) production, which damaged mitochondrial DNA (mtDNA) and ultimately led to apoptosis in INS-1 cells. To firmly establish the link between FFA-generated mtDNA damage and apoptosis, we stably transfected INS-1 cells with an expression vector containing the gene for the DNA repair enzyme human 8-oxoguanine DNA glycosylase/apurinic lyase (hOGG1) downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase. Successful integration of MTS-OGG1 into the INS-1 cellular genome was confirmed by Southern blot analysis. Western blots and enzyme activity assays revealed that hOGG1 was targeted to mitochondria and the recombinant enzyme was active. MTS-OGG1 cells showed a significant decrease in FFA-induced mtDNA damage compared with vector-only transfectants. Additionally, hOGG1 overexpression in mitochondria decreased FFA-induced inhibition of ATP production and protected INS-1 cells from apoptosis. These results indicate that mtDNA damage plays a pivotal role in FFA-induced beta-cell dysfunction and apoptosis. Therefore, targeting DNA repair enzymes into beta-cell mitochondria could be a potential therapeutic strategy for preventing or delaying the onset of type 2 diabetes symptoms.
Rachek
Lyudmila I
LI
Department of Cell Biology and Neuroscience, College of Medicine, University of South Alabama, Mobile, AL 36688, USA. lrachek@jaguar1.usouthal.edu
Thornley
Nancy P
NP
Grishko
Valentina I
VI
LeDoux
Susan P
SP
Wilson
Glenn L
GL
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D004249:DNA Damage; D053938:DNA Fragmentation; D045647:DNA Glycosylases / Q000172:deficiency / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000201:enzymology; D051381:Rats; D014162:Transfection
D004272:DNA, Mitochondrial; D005230:Fatty Acids, Nonesterified; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human