May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Iron Influences NADPH Production by Both Isocitrate Dehydrogenase and Glucose-6-phosphate Dehydrogenase
Author Affiliations & Notes
  • M. McGahan
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, United States
  • L.N. Fleisher
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, United States
  • M. Goralska
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, United States
  • Footnotes
    Commercial Relationships  M. McGahan, None; L.N. Fleisher, None; M. Goralska, None.
  • Footnotes
    Support  EY-04900
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1244. doi:
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      M. McGahan, L.N. Fleisher, M. Goralska; Iron Influences NADPH Production by Both Isocitrate Dehydrogenase and Glucose-6-phosphate Dehydrogenase . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1244.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: There are three major enzymes involved in NADPH production in the cytoplasm, cytoplasmic isocitrate dehydrogenase (c-ICD), malic enzyme and glucose-6-phosphate dehydrogenase (G-6-PD). The substrate for c-ICD is derived from aconitase production of isocitrate. Cytoplasmic aconitase is an Fe dependent enzyme and it has been proposed that Fe could regulate the production of NADPH by this pathway. In the present study we examined the effects of Fe depletion on the production of NADPH by all three pathways in cultured canine lens epithelial cells (LEC). Methods: Primary cultures of LEC were grown in DMEM+10% fetal calf serum, these cells are Fe replete. In one experiment, some LEC were placed in low Fe medium which also contained the Fe chelator, desferrioxamine. Control LEC were maintained in the Fe replete medium. The LEC were incubated for 24 hours. In another set of experiments, the medium of half of the Fe depleted cells was changed back to the Fe replete medium after 24h depletion, the other half were maintained under Fe depleted conditions. These two sets of cells were incubated for an additional 24h. At the end of the treatment periods, the cells were lysed and enzymatic production of NADPH by each pathway was measured spectrophotometrically (units are nmoles NADPH/mg pr/min in all cases). Results: In the first set of experiments, Fe depletion resulted in an almost 90% decrease in NADPH production by c-ICD (from 0.9+/-0.2 to 0.12+/-0.03). NADPH production by malic enzyme and G-6-PD was unaffected. Addition of Fe replete mediua back to Fe starved cells resulted in an increase in NADPH production via c-ICD, again malic anzyme and G-6-PD were unaffected. However, in the remaining cells, after two days of Fe depletion and DFO exposure, cytoplasmic c-ICD production of NADPH was still significantly depressed. In contrast, production of NADPH by G-6-PD was more than doubled (11.6+/-2.3 vs. 26.2+/-1.8). Conclusions: Not surprisingly, the production of NADPH by c-ICD is reduced by Fe depletion. This is likely due to decreased availability of its substrate as a result of decreased activity of aconitase (an Fe dependent enzyme). However, the finding of increased G-6-PD activity after two days of Fe depletion indicates that these pathways are interdpendent and that Fe may play an important role in regulation of NADPH production in these cells, and not exclusively via the c-ICD pathway.

Keywords: oxidation/oxidative or free radical damage • antioxidants • metabolism 
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