June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Glutamine is an essential contributor to the human corneal endothelial ATP pool
Author Affiliations & Notes
  • Wenlin Zhang
    School of Optometry, Indiana University Bloomington, Bloomingotn, IN
  • Diego G Ogando
    School of Optometry, Indiana University Bloomington, Bloomingotn, IN
  • Joseph A Bonanno
    School of Optometry, Indiana University Bloomington, Bloomingotn, IN
  • Footnotes
    Commercial Relationships Wenlin Zhang, None; Diego Ogando, None; Joseph Bonanno, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2578. doi:
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      Wenlin Zhang, Diego G Ogando, Joseph A Bonanno; Glutamine is an essential contributor to the human corneal endothelial ATP pool. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2578.

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

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Abstract

Purpose: Glutamine has long been viewed as an essential nutrient in corneal graft storage and corneal endothelial (CE) culture medium. We recently characterized the highly expressed CE membrane protein SLC4A11 as a novel ammonia transporter, suggesting active glutamine metabolism and the net production of ammonia. We hypothesized CE cells can utilize glutamine as an alternative carbon source for ATP production, which potentially is an essential substrate supporting CE Na-K-ATPase and ‘pump’ function.

Methods: Enzymes and transporters required for glutamine metabolism were screened by RT-PCR in human CE specimens collected from eye bank tissue. Glutamine consumption in an immortalized human CE cell line was measured by ammonia release into culture medium by colorimetric assay. ATP production by CE in glucose only (5 g/l), glutamine only (4 mM), both or neither substrate was measured by luciferin-luciferase assay in human CE cell lines. All data are expressed as mean ± s.e.m.

Results: We detected the expression of the key enzyme in glutamine metabolism in human CE: phosphate-dependent glutaminase, which catalyzes the first step in glutamine oxidation. Both kidney isoform glutaminase 1 (GLS1) and liver isoform (GLS2) are expressed (GLS1>GLS2). Excitatory Amino Acid Transporter 1-3 (EAAT 1-3), associated with glutamine metabolism, were also detected with expression level EAAT3≈EAAT2>EAAT1. Ammonia release (mM) was dose dependent on glutamine concentration (n=6 at each concentration, 0.1, 0.5, 1, 2, 4 mM) with highest at 4 mM (1.14±0.01) and lowest at 0.1 mM (0.74±0.02) (Independent t-test, p<0.001). Ammonia levels can be fitted against glutamine concentration with the Michaelis-Menten equation (R2=0.918, p<0.0001), and yield a Km of 1.99±0.62 mM (p=0.0034), similar to previously reported glutaminase Km 2~5 mM. ATP production (nmol/106 cells) was highest in glucose and glutamine combined medium (1.10±0.09), slightly lower in glutamine only medium (1.00±0.12), and significantly lower in glucose only (0.52±0.07) and no substrate medium (0.34±0.07). (n=23 in each condition, One-way ANOVA, P<0.001).

Conclusions: Human CE can actively use glutamine as an essential carbon source for ATP production through glutaminase. This work could provide insight in optimizing cornea graft storage medium and the pathophysiology caused by mutated ammonia transporter SLC4A11.

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