May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Glucose, Not Lactate, is the Preferred Substrate Metabolized by Cultured Retinal Cells
Author Affiliations & Notes
  • B.S. Winkler
    Eye Research Institute, Oakland University, Rochester, MI, United States
  • C.A. Starnes
    Eye Research Institute, Oakland University, Rochester, MI, United States
  • M.W. Sauer
    Eye Research Institute, Oakland University, Rochester, MI, United States
  • Z. Firouzgan
    Eye Research Institute, Oakland University, Rochester, MI, United States
  • S. Chen
    Eye Research Institute, Oakland University, Rochester, MI, United States
  • Footnotes
    Commercial Relationships  B.S. Winkler, None; C.A. Starnes, None; M.W. Sauer, None; Z. Firouzgan, None; S. Chen, None.
  • Footnotes
    Support  NIH Grant EY 10015
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4573. doi:
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      B.S. Winkler, C.A. Starnes, M.W. Sauer, Z. Firouzgan, S. Chen; Glucose, Not Lactate, is the Preferred Substrate Metabolized by Cultured Retinal Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4573.

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

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Abstract

Abstract: : Purpose: Glucose has long been considered the substrate for retinal energy metabolism . Recently, an alternative hypothesis (Metabolic Coupling) suggests that neuronal metabolism depends on the lactate produced from Muller cells. This hypothesis is examined from the perspective of substrate availability and enzyme kinetics in cultured retinal neuronal and glial cells. Methods: Cultured rat Muller cells (rMC-1), human retinal pigment epithelial cells (RPE) and transformed photoreceptor cells (661W) were each grown to confluency under standard conditions. Aerobic and anaerobic lactic acid production were measured during incubation of the cells in media containing 5.5 mM glucose or 5.5 mM glucose and 10 mM lactic acid +/- Antimycin A to inhibit mitochondria. After 4 hr incubations, cellular ATP content was measured. The dependence of the activity of lactic acid dehydrogenase (LDH) in cytosolic extracts on the concentrations of pyruvate (forward reaction) and lactate (backward reaction) was evaluated together with PAGE of the isoform patterns in the rMC-1, RPE and 661W cells; these activity profiles and PAGE bands were compared to those of purified LDH1 (heart) and LDH5 (muscle). Results: All cell types incubated with 5.5 mM glucose produced lactate aerobically and anaerobically at linear rates, the anaerobic rate being 2-3-fold higher. Cells incubated with 5.5 glucose and 10 mM lactate produced lactate aerobically and anaerobically at the same rates as cells incubated with glucose alone. In rMC-1 and RPE cells, anaerobic ATP content was similar to aerobic ATP content, but in 661W cells anaerobic ATP content was about 70% of the aerobic content. In rMC-1 cells, the PAGE and kinetic studies revealed that LDH5 was the predominant, if not sole, isoform. RPE and 661W cells expressed multiple isoforms, with muscle-type isoforms predominating. Consistent with the PAGE data, kinetic studies in RPE and 661W cells showed that the Kms (4 mM lactate) for the backward reaction were similar to the Km for LDH5 (4 mM) but not for LDH1 (1 mM). The Kms for the forward reaction in the cells fell between the Km values for LDH1 (0.05 mM pyruvate) and LDH5 (0.25 mM), indicating contributions from multiple isoforms. Conclusions: The results show that cultured rMC-1, RPE and 661W cells produce lactate aerobically and anaerobically, even in the presence of a high starting ambient concentration of lactate (10 mM). Despite a differential distribution of LDH isoforms in the cells, a net production of lactate is observed. Results suggest that RPE and photoreceptor cells use glucose, and not glial derived lactate, as their major substrate.

Keywords: retina • metabolism • retina: neurochemistry 
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