October 2008
Volume 49, Issue 10
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Retinal Cell Biology  |   October 2008
Nuclear Magnetic Resonance and Biochemical Measurements of Glucose Utilization in the Cone-Dominant Ground Squirrel Retina
Author Affiliations
  • Barry S. Winkler
    From the Eye Research Institute and the
  • Catherine A. Starnes
    From the Eye Research Institute and the
  • Brandon S. Twardy
    From the Eye Research Institute and the
  • Diane Brault
    Department of Chemistry, Oakland University, Rochester, Michigan.
  • R. Craig Taylor
    Department of Chemistry, Oakland University, Rochester, Michigan.
Investigative Ophthalmology & Visual Science October 2008, Vol.49, 4613-4619. doi:10.1167/iovs.08-2004
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      Barry S. Winkler, Catherine A. Starnes, Brandon S. Twardy, Diane Brault, R. Craig Taylor; Nuclear Magnetic Resonance and Biochemical Measurements of Glucose Utilization in the Cone-Dominant Ground Squirrel Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(10):4613-4619. doi: 10.1167/iovs.08-2004.

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

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Abstract

purpose. To provide quantitative information on glucose utilization in cone-dominant ground squirrel retinas.

methods. Ground squirrel eyecups were incubated in medium containing 14C-glucose, and the production of 14CO2 was measured. Measurements were also made of lactic acid production (glycolysis). Nuclear magnetic resonance (NMR) was used to track metabolites generated from 13C-1 glucose.

results. Ground squirrel eyecups produced lactate at a high rate and exhibited normal histology. Light-adaptation reduced glycolysis by 20%. Ouabain decreased glycolysis by 25% and decreased 14CO2 production by 60%. Blockade of glutamate receptors had little effect on the glycolysis and 14CO2 produced. When metabolic responses were restricted to photoreceptors, light caused a 33% decrease in 14CO2 production. The rate of 14CO2 production was less than 10% of lactate production. Lactate was the major product formed from 13C-glucose. Other 13C-labeled compounds included glutamate, aspartate, glutamine, alanine, taurine, and GABA. Lactate was the only product detected in the medium bathing the ground squirrel retinas. The rod-dominant rat retina exhibited a similar pattern of metabolites formed from glucose.

conclusions. Lactate, not CO2, is the major product of glucose metabolism in both ground squirrel and rat retinas. Active Na+ transport, however, depends more on ATP produced by mitochondria than by glycolysis. A relatively high fraction of ATP production from glycolysis and glucose oxidation continues in the absence of active Na+ pumping and glutamatergic transmission. Major neurotransmitters are synthesized from the aerobic metabolism of glucose; anoxia-induced impairment in retinal synaptic transmission may be due to depletion of neurotransmitters.

Nearly all studies to date of energy metabolism in mammalian retinas have been performed on rod-dominant retinas that possess very few cones. 1 2 3 4 5 Because of this, measurements of the rates of aerobic and anaerobic lactic acid production, mitochondrial utilization of substrates, and respiration in these retinas in darkness and in light reflect rod-dependent activities, the contribution from cones being too small to assess reliably. Achieving the goal of providing quantitative information on cone-dependent energy metabolism requires that we have good animal models. To this end, the cone-dominant ground squirrel (Spermophilus tridecemlineatus) retina that contains approximately 85% to 95% cones is a good choice. 6 7 Accordingly, we report measurements of parameters of glucose metabolism in this cone-dominant retina and compare these data with those previously obtained in rod-dominant retinas that contain few cones (Sprague-Dawley, Rattus norvegicus). 2  
Materials and Methods
Ground squirrels were trapped live in the spring and summer within a radius of 150 miles from Chicago (TLS Research, Bloomingdale, IL). They were delivered to Oakland University in mid-July. Squirrels were caged individually, provided with nesting material (shredded paper), and given ordinary rodent chow and water. They were housed in a climate-controlled room on a 12-hour light–12-hour dark cycle for at least 2 weeks before use. Experiments were conducted between August 1 and September 15. Ground squirrels are active during this time of year; hence, prehibernation or hibernation 8 is not a confounding factor in this study. Although it is nearly impossible to know with certainty the age of wild-trapped ground squirrels, TLS management indicated that the ground squirrels were likely to be between 1 and 3 years of age and thus were mature. Eyecups were obtained from dark- and light-adapted (ordinary room light exposure) ground squirrels between 10 AM and 12 PM after carbon dioxide asphyxiation and removal of anterior eye structures. The retinas were also obtained from 3-month-old adult rats (Harlan Sprague-Dawley, Indianapolis, IN) by procedures that have been described previously. 2 Briefly, the rat eye was proptosed by placing forceps around the optic nerve near its exit from the eye. The eye was transected along the equator, and the cornea and lens were removed. With gentle upward movement of the forceps, the retina was detached completely from the RPE. The entire isolated retina was then deposited in a Petri dish filled with incubation medium. All animals used in the experiments were treated and maintained in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the study was approved by the Oakland University Institutional Animal Care and Use Committee. 
Ground squirrel eyecups were prepared by removal of anterior structures from eyes enucleated from dark-adapted animals under dim red light. The tissues were then incubated in continuous darkness except for brief exposures to dim red light (∼10 seconds every 30 minutes) for sample taking during the course of an experiment. We refer to this condition as dark-adapted, even though the intermittent exposure to red light could briefly activate red-sensitive cones. Eyecups were also prepared in the presence of ordinary room light and incubations conducted in the presence of continuous light. This is referred to as the light-adapted condition, although it is possible that in these cone-dominant retinas, the amount of light may not be enough to produce maximum changes in the metabolic parameters. For measurement of lactic acid production, the eyecups were added to flasks (1 eyecup/flask) containing 10 mL of bicarbonate-buffered, oxygenated (95% O2/5% CO2) medium of normal ionic composition and containing 10 mM glucose as the sole exogenous substrate (control medium). The pH was 7.4, temperature was maintained at 37°C, and gassing was continuous in a gentle stream. Chemical inhibition of mitochondrial electron transport was achieved by addition of 10−5 M antimycin A to the medium (referred to in the text as the anaerobic condition). Estimates of lactic acid production were obtained by removing 100 μL from the incubation medium every 30 minutes. Lactate was measured with an LDH-based reaction kit 2 (Sigma-Aldrich, St. Louis, MO). The extent to which glucose was oxidized by mitochondria was assessed by measurements of the appearance of 14CO2 after incubation of the ground squirrel eyecups in medium containing 14C-3,4 glucose (1 eyecup/10 mL), as described in detail previously. 9 Labeling of glucose carbons in positions 3 and 4 reflects the metabolic production of CO2 at the step of pyruvate decarboxylation, the entry of pyruvate into the Krebs cycle. Ouabain (10−3 M) was added to medium to inhibit ATP-dependent, active Na+/K+ ion transport. 10 Glutamatergic transmission was blocked with a combination of 0.05 mM CNQX (inhibitor of kainate and quisqualate receptors), 0.01 mM MK-801 (inhibitor of NMDA receptors), and 0.1 mM APB (inhibitor of metabotropic glutamate receptors on ON-bipolar cells). 11 The data for lactic acid production and mitochondrial glucose oxidation are presented as the mean ± SD. Results were analyzed by unpaired Student’s t-test, and P < 0.05 was considered to be statistically significant. 
To determine the metabolites produced from 13C-1 glucose in tissue extracts and in medium, 13C-NMR spectra were obtained with a nuclear magnetic resonance spectrometer (Avance 200; Bruker, Newark, DE). After incubation for the periods described in the Results section, ground squirrel retinas were washed briefly with ice-cold unlabeled medium, homogenized in 0.7 mL cold 5% perchloric acid in 20% D2O (two ground squirrel retinas were pooled), and centrifuged at 14,000 rpm for 15 minutes. Exactly 0.7 mL of the supernatant was used for each NMR sample. A similar procedure was used for NMR spectroscopy of incubated rat retinas, but in this case, eight retinas were homogenized. For NMR analysis of metabolites in the incubation medium, 0.6 mL samples were added directly to 0.1 mL 20% D2O. Dioxane was included as an internal standard. Quantitative 13C-NMR spectra were obtained by using the inverse-gated decoupling method that produces 1H-decoupled 13C spectra without nuclear Overhauser enhancement (NOE). 12 13 Therefore, the intensities of the individual peaks are related to the amount of compound present in the samples. Typically, the number of scans was 11,500 with a 4-second delay between each scan. 
Light and electron microscopic examination of selected retinas were performed as previously described. 14 Briefly, tissues were fixed in a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2.5 hours at room temperature. After they were washed in buffer and postfixed in 0.1 M cacodylate-buffered 1% osmium tetroxide for 3 hours at room temperature, the tissues were stained overnight in cold 2% solution of uranyl acetate in 0.05 M maleate buffer, dehydrated, and embedded in an Epon-Araldite mixture. Semithin and ultrathin sections were cut on an ultramicrotome. Semithin sections were stained with toluidine blue, and the ultrathin sections were stained with lead citrate and examined by electron microscope (model 300; Phillips, Cincinnati, OH). 
Results
The histology of ground squirrel eyecups was examined after incubation of the tissues for 2 hours in control medium. As shown in the top micrograph of Figure 1 , the cytologic features of the retina were well-maintained under the control condition. The electron micrographs at the bottom of Figure 1compare the density of mitochondria in cone inner segments of ground squirrel retinas with the density of these organelles in rat rod inner segments. As shown, mitochondria are substantially more densely packed in cone inner segments. 
Table 1provides information on the rate of appearance of lactic acid into the medium bathing dark- and light-adapted ground squirrel retinas. In comparison to the rate of lactic acid production in darkness (4.5 ± 0.4 micromoles/eyecup/hour), the rate in light-adapted retinas was decreased by 18%. Under anaerobic conditions, the rates of glycolysis in darkness and in light, respectively, were increased by 40% and 50%, that is, a similar Pasteur effect was observed. When the eyecups were incubated in glucose-free medium, aerobic lactic acid production declined rapidly to a low level, and production ceased after approximately 1 hour; the total amount produced aerobically amounted to 1 micromole/eyecup (data not shown). Incubation of ground squirrel eyecups in medium containing the cocktail of chemicals (CNQX, MK-801, and APB) designed to inhibit glutamatergic neurotransmission in the outer and inner plexiform layers had no significant effect on the rate of aerobic lactic acid production in darkness and in light. Ouabain decreased aerobic lactate production in darkness and in light, respectively, by 27% and 24%. 
The data in Table 2provide information on the rate of mitochondrial glucose oxidation in ground squirrel retinas. The rate of glucose oxidation was not significantly different in dark- and light-adapted retinas. The cocktail of inhibitors of glutamate receptors had no effect on glucose oxidation in darkness, but decreased glucose oxidation in light-adapted retinas by 33%. Incubation of ground squirrel retinas in medium containing ouabain decreased glucose oxidation by 60% and 67%, respectively, in darkness and in light. 
From the quantitative measurements of aerobic lactic acid production and mitochondrial glucose oxidation presented in Tables 1 and 2 , estimates of the fraction of glucose that was metabolized by these two pathways in darkness and in light were derived. The results of these calculations are shown in Table 3 . The data reveal that aerobic glycolysis accounts for the overwhelming (>90%) fraction of glucose used in the cone-dominant ground squirrel retina in darkness and in light. 
13C-NMR spectra of extracts of ground squirrel retinas obtained after a 2-hour incubation of dark-adapted tissues in control medium containing labeled glucose are reported in Figure 2(top, low resolution; bottom, high resolution). It is clear that the overwhelming incorporation of 13C-1 glucose was into lactate, with substantially lower rates of incorporation into amino acids (alanine, aspartate, GABA, glutamate, glutamine, and taurine). Of the various labeled amino acids, glutamate showed the highest incorporation. The two C-1 resonances of the α-and β-anomers of glucose were also present. Figure 3shows that lactate was the only labeled product detected in the medium (low resolution) bathing the ground squirrel retinas. 
13C-NMR spectra obtained from extracts of light-adapted ground squirrel retinas and of the medium bathing these retinas were similar to the spectra shown in Figures 2 and 3for dark-adapted tissues. 
For purposes of comparison with a rod-dominant retina, 13C-NMR spectra were obtained from extracts of dark-adapted rat retinas that were incubated in the same conditions as used in the incubations of the ground squirrel retinas. As shown in Figure 4(top and bottom), in comparison to the ground squirrel retinas a similar pattern of incorporation of 13C-1 glucose was found in rat retinas: highest incorporation into lactate; much lower incorporation into amino acids. Glutamate was the highest labeled amino acid. As was the case for the ground squirrel retinal incubations, lactate was the only labeled product detected in the medium bathing the isolated rat retinas (Fig. 5)
Discussion
From the time that anatomic descriptions of the retinas of diurnal squirrels showed that these tissues contained predominantly cone photoreceptor cells (∼95% of the total photoreceptor cells), 6 7 there has been considerable interest in using these retinas to uncover cone-dependent visual mechanisms. Wide-ranging electrophysiological and biochemical studies of these mechanisms have been performed and the results compared to the behavior observed in rod-dominant retinas. Included are studies on electrophysiological responses of photoreceptor cells, 15 spectral sensitivity measurements, 16 17 expression of phototransduction genes, 18 effects of retinal detachment and reattachment, 19 20 and application of chemical poisons. 21  
It is known that, in primates, mitochondria comprise “74% to 85% of cone ellipsoids and 54% to 66% of rod ellipsoids,” and “per unit volume of outer segment, cones contain ten times as much mitochondria as rods.” 22 Figure 1(bottom) shows that cone inner segments in the ground squirrel retina had a much higher density of mitochondria than did the rod inner segments in the rat retina. As in all cells, the mitochondria supply high-energy phosphates in the form of ATP and GTP that fuel many of the energy-consuming processes in the photoreceptor cell. The fact that cone inner segments have more mitochondria than do rod inner segments has led to the suggestion that the energy needs of cones are greater than in rods. 23 24 Support for this suggestion has come from comparative structural analyses of the three-dimensional cytoarchitecture of mitochondria and their cristae in cones and rods. 23 24 On the other hand, it has been suggested that the dense packing of mitochondrial membranes in cone inner segments results in a higher optical index of refraction (relative to rod inner segments), thus serving to better direct the flow of photons into outer segments (i.e., to enhance waveguide properties). 22 To date, there have been no reports on the metabolic properties of ground squirrel retinas, which serve as a representative model of cone-dominant mammalian retinas. The present study was therefore undertaken to provide quantitative measurements on glucose utilization and mitochondrial metabolism in the ground squirrel retina and to compare these measurements with those made previously on rod-dominant retinas. Particular attention was directed toward assessing the relative contributions of active ion transport and glutamatergic neurotransmission on the rates of glycolysis and mitochondrial glucose oxidation. 
Rod-dominant mammalian retinas exhibit two reproducible and consistent metabolic properties: They convert glucose to lactic acid at a high rate, and the formation of lactic acid is rapid even in the presence of oxygen (referred to as aerobic glycolysis or aerobic lactic acid formation). 1 2 4 5 It is clear from the data in Table 1that cone-dominant retinas possess similar properties. High rates of aerobic glycolysis were observed in dark- and light-adapted retinas, the rate in light being only 20% less than in the dark. This result suggests that in the presence of oxygen, a portion of the energy required for maintenance of the dark current in the ground squirrel is derived from glycolysis. This contrasts with results obtained in rod-dominant rabbit and rat retinas where interruption of the dark current by steady light under control conditions caused no significant change in the rate of aerobic glycolysis. 2 4 11 Glycolytically produced ATP is used by the Na+,K+-ATPase, since incubation of ground squirrel retinas in medium containing 1 mM ouabain led to a 25% decline in the rate of aerobic glycolysis in both dark- and light-adapted retinas. To examine the effects on lactate production after blockade of glutamatergic synaptic transmission at NMDA and non-NMDA receptors, a cocktail of inhibitory glutamate analogues was used (CNQX, MK-801, and APB). Exposure of retinas to medium containing this cocktail of chemicals in either darkness or light had no significant effect on the rate of aerobic glycolysis relative to the control rate. This result differs from that reported for dark-adapted rabbit retinas, in which lactate acid production was decreased by 44% after incubation of the retina in medium containing a similar combination of inhibitory glutamate analogues. 11  
The data in Table 2show that there were no significant differences between the rates of glucose oxidation in dark- and light-adapted ground squirrel retinas incubated in normal medium (i.e., in medium lacking the glutamate receptor blockers). Addition of the cocktail of glutamatergic chemicals to medium bathing dark-adapted ground squirrel eyecups had no effect on 14CO2 production. However, when metabolic responses to the dark-light shifts were restricted to photoreceptors as a consequence of blocking synaptic transmission between photoreceptors and second-order neurons, steady light caused a 33% decrease in mitochondrial glucose oxidation (e.g., 0.08 vs. 0.12 micromoles 14CO2/eyecup/hr. This difference between glucose oxidation in dark and light indicates that light decreases photoreceptor cell mitochondrial activity, as it does in other species. 3 25 26 27 28 The magnitude of this light-induced decrease in mitochondrial oxidation in a cone-dominant retina is similar to that reported for the effects of light on oxygen consumption in the perifoveal region of macaque retinas. 28 In that study, it was also reported that oxygen consumption in the fovea decreased under light-adapted conditions, but by a smaller amount than the decrease (28%) observed in the perifoveal region. Surprisingly, perifoveal oxygen consumption was higher than the consumption of oxygen in the fovea, despite the fact that “the mitochondrial density in foveal cone inner segments is 60% higher than that in perifoveal rods and the inner segments are longer in the fovea.” 28  
The data in Table 2also provide some information regarding light-induced changes in inner retinal metabolism in a cone-dominant retina. That light decreased the rate of 14CO2 production in cocktail-treated retinas, but not in untreated retinas, suggests that inner retinal 14CO2 must have increased in light to offset the decrease in 14CO2 produced in photoreceptors. The underlying assumption is that in the presence of the cocktail of inhibitors of glutamatergic transmission, inner retinal metabolism is the same in dark and light. In rod-dominant retinas, including rat 25 and cat, 29 inner retinal metabolism is the same in darkness and constant light, as evaluated by different methods. The explanation for this difference in the effect of light on inner retinal metabolism between cone-dominant and rod-dominant retinas is not known. 
The largest decline in mitochondrial glucose oxidation was observed after incubation of ground squirrel retinas in medium containing ouabain (60%–67% decrease). The concentration used (1 mM) is 10-fold greater than that needed to completely inhibit the Na+,K+-ATPase in isolated rat retinas. 10 This result indicates that a major fraction of ATP produced by the mitochondria in the ground squirrel retina supports active Na+ transport. Nevertheless, a relatively high fraction of ATP production from mitochondrial activity (33%–40%) continues in the absence of active Na+ pumping and glutamatergic transmission in the inner retina. A similar finding was reported for the isolated rabbit retina 4 11 —that is, about half of the total energy generated by the retina could not be linked to active Na+ transport or glutamate-dependent synaptic transmission. 
Table 3provides estimates of the fraction of glucose that was metabolized, respectively, by aerobic glycolysis and mitochondrial glucose oxidation. The calculations were derived straightforwardly: 1 mole of glucose yields 2 moles of lactic acid, and the number of moles of 14CO2 produced from the oxidation of 14C-3,4 glucose divided by 2 equals the number of moles of glucose oxidized to CO2. The total glucose used is the sum of the glucose glycolyzed and the glucose oxidized (but, see later discussion of NMR data). In the cone-dominant ground squirrel retina, aerobic glycolysis thus appears to account for 94% to 96% of the total glucose used aerobically. This shows that the major fraction of glucose is converted to lactic acid in the ground squirrel retina, a conclusion in agreement with that found previously in rod-dominant cat, rat, and rabbit retinas. 1 2 4 5 Since glycolysis is 18-fold less efficient than mitochondria in producing ATP per mole of glucose used, the small number of glucose molecules that are oxidized contribute nearly 50% of the total ATP production in the ground squirrel retina, the other 50% coming from glycolysis. 
The 13C-NMR data provide critical, new information on the metabolism of glucose in cone-dominant (Figs. 2 3)and rod-dominant (Figs. 4 5)retinas. First, the NMR results reinforce the biochemical measurements showing that lactic acid is the predominant product of glucose metabolism. This finding was true whether the NMR was performed on extracts of the retina or on samples taken from the incubation medium bathing the tissue. In both cases, accumulation of label from 13C-1 glucose was greatest in lactate. Furthermore, lactate was the only labeled compound detected in the incubation medium under the conditions of the experiments and the sensitivity of the NMR system. Second, NMR analysis of retinal extracts showed clearly that glucose was converted to many compounds besides lactate, most of these being readily recognized as excitatory and inhibitory amino acid transmitters such as l-glutamic acid, l-aspartic acid, and GABA. In addition, glucose was metabolized to taurine and l-glutamine, the former representing activity occurring in photoreceptor cells, within which taurine is in very high concentration, and the latter representing activity residing in Müller cells, the cellular site of glutamine synthetase. 30 31 A comparison between the 13C-NMR labeling patterns in the ground squirrel retina (Figs. 2 3)with those in the rat retina (Figs. 4 5)reveals a similar picture. 
In terms of the pecking order of compounds formed from 13C-1 glucose in ground squirrel and rat retinas, lactate was the most abundant product, followed by l-glutamate. This finding suggests that glucose metabolism has an essential role, not just in ATP production from glycolysis (lactate production) and from respiration (CO2 formation), but also in the maintenance of the excitatory neurotransmitter used by rod and cone photoreceptor cells and by bipolar cells. Since the formation of glutamate and other amino acids from glucose depends on mitochondrial enzymes that catalyze transamination (e.g., aspartate aminotransaminase) and dehydrogenase (e.g., glutamate dehydrogenase) reactions, it is expected that inhibition of mitochondrial activity would lead to a decrease in the levels of these amino acids and inhibition of synaptic transmission. In this regard, it is of particular interest that incubation of dark-adapted isolated rat retinas in glucose-containing medium equilibrated with 95% N2/5% CO2 (no oxygen) results in loss of the light-induced b-wave of the ERG, but the a-wave (photoreceptor potential) continues to be generated at a near-normal level. 32 The reason for the selective loss of the b-wave during anoxia is unclear. One possibility is that anoxia causes a decrease in ATP production and a consequent decline in utilization by ATP-dependent processes in ON-bipolar cells that contribute directly to the generation of the b-wave, 33 thereby compromising their physiological responsiveness. However, loss of the b-wave occurs before there is a significant decline in retinal ATP. 2 32 Since the generation of the b-wave depends on the light-dependent modulation of synaptic release of glutamate from photoreceptor cells to ON-bipolar cells, an impairment of the synaptic machinery (i.e., glutamate depletion due to anoxia) would result in a decline or a loss of the b-wave. Given that the formation of glutamate depends on the mitochondrial (aerobic) metabolism of glucose, it is reasonable to suggest that anoxia would decrease the formation of glutamate (and other amino acids) from glucose. A decrease in the formation of glutamate would be expected to result in a decline or depletion of its concentration throughout the photoreceptor cell, including the synaptic terminal. If this were to occur, photoreceptor synaptic transmission would be impaired, and the amplitude of the b-wave would decline. Depletion of glutamate in retinal cells in response to anoxia would also be expected to reduce its availability for conversion to glutamine via glutamine synthetase in Müller cells. 30 31 Glutamine levels would then decline as well, disrupting the potential for recycling of glutamate (glutamate-to-glutamine cycle) back into the photoreceptor synaptic vesicles. 34  
 
Figure 1.
 
Top: representative light micrograph of a semithin section of ground squirrel retina incubated in normal medium for 2 hours; bottom: electron micrographs through outer and inner segments of rat rods (left) and ground squirrel cones (right). Pigment surrounding the outer segments in the ground squirrel is representative of the apical processes of the RPE.
Figure 1.
 
Top: representative light micrograph of a semithin section of ground squirrel retina incubated in normal medium for 2 hours; bottom: electron micrographs through outer and inner segments of rat rods (left) and ground squirrel cones (right). Pigment surrounding the outer segments in the ground squirrel is representative of the apical processes of the RPE.
Table 1.
 
Lactate Production in Ground Squirrel Retinas*
Table 1.
 
Lactate Production in Ground Squirrel Retinas*
Condition Inhibitors Aerobic Anaerobic Pasteur Effect
Dark None 4.5 ± 0.4 (9) 6.1 ± 0.3 (5) 1.4
Light None 3.7 ± 0.4 (8) 5.4 ± 0.3 (5) 1.5
Dark Cocktail 4.5 ± 0.5 (6)
Light Cocktail 3.5 ± 0.7 (5)
Dark Ouabain 3.3 ± 0.3 (8)
Light Ouabain 2.8 ± 0.5 (4)
Table 2.
 
14CO2 Production from 14C-3,4 Glucose in Ground Squirrel Retinas*
Table 2.
 
14CO2 Production from 14C-3,4 Glucose in Ground Squirrel Retinas*
Condition Inhibitors 14CO2
Dark None 0.11 ± 0.04 (5)
Light None 0.12 ± 0.05 (5)
Dark Cocktail 0.11 ± 0.04 (3)
Light Cocktail 0.08 ± 0.04 (3)
Dark Ouabain 0.04 ± 0.01 (4)
Light Ouabain 0.04 ± 0.01 (4)
Table 3.
 
Quantitative Estimates of Aerobic Glucose Utilization by Glycolysis and Mitochondrial Oxidation in Ground Squirrel Retinas*
Table 3.
 
Quantitative Estimates of Aerobic Glucose Utilization by Glycolysis and Mitochondrial Oxidation in Ground Squirrel Retinas*
Dark Light
Lactate Production 4.50 3.70
Glucose-to-lactate 2.25 1.85
Glucose oxidized by mitochondria 0.06 0.06
Glucose glycolyzed as a % of total utilized 96 94
Figure 2.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of dark-adapted ground squirrel retinas incubated for 2 hours in medium containing 13C-1 glucose. Inset: metabolites that appear between 25 and 35 ppm. Glu, glutamate; Tau, taurine; GABA, γ-aminobutyric acid; Gln, glutamine.
Figure 2.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of dark-adapted ground squirrel retinas incubated for 2 hours in medium containing 13C-1 glucose. Inset: metabolites that appear between 25 and 35 ppm. Glu, glutamate; Tau, taurine; GABA, γ-aminobutyric acid; Gln, glutamine.
Figure 3.
 
The 13C NMR spectrum (low resolution) of metabolites from D2O extracts of the medium bathing dark-adapted ground squirrel retinas incubated for 2 hours in medium containing 13C-1 glucose.
Figure 3.
 
The 13C NMR spectrum (low resolution) of metabolites from D2O extracts of the medium bathing dark-adapted ground squirrel retinas incubated for 2 hours in medium containing 13C-1 glucose.
Figure 4.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of dark-adapted rat retinas incubated for 2 hours in medium containing 13C-1 glucose. Inset: metabolites that appear between 25 and 35 ppm. Glu, glutamate; Tau, taurine; GABA, γ-aminobutyric acid; Gln, glutamine.
Figure 4.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of dark-adapted rat retinas incubated for 2 hours in medium containing 13C-1 glucose. Inset: metabolites that appear between 25 and 35 ppm. Glu, glutamate; Tau, taurine; GABA, γ-aminobutyric acid; Gln, glutamine.
Figure 5.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of the medium bathing dark-adapted rat retinas incubated for 2 hours in medium containing 13C-1 glucose.
Figure 5.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of the medium bathing dark-adapted rat retinas incubated for 2 hours in medium containing 13C-1 glucose.
The authors thank Loan Dang for the histology and electron microscopy and Mary Duenow for assistance in preparation of the figures. 
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Figure 1.
 
Top: representative light micrograph of a semithin section of ground squirrel retina incubated in normal medium for 2 hours; bottom: electron micrographs through outer and inner segments of rat rods (left) and ground squirrel cones (right). Pigment surrounding the outer segments in the ground squirrel is representative of the apical processes of the RPE.
Figure 1.
 
Top: representative light micrograph of a semithin section of ground squirrel retina incubated in normal medium for 2 hours; bottom: electron micrographs through outer and inner segments of rat rods (left) and ground squirrel cones (right). Pigment surrounding the outer segments in the ground squirrel is representative of the apical processes of the RPE.
Figure 2.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of dark-adapted ground squirrel retinas incubated for 2 hours in medium containing 13C-1 glucose. Inset: metabolites that appear between 25 and 35 ppm. Glu, glutamate; Tau, taurine; GABA, γ-aminobutyric acid; Gln, glutamine.
Figure 2.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of dark-adapted ground squirrel retinas incubated for 2 hours in medium containing 13C-1 glucose. Inset: metabolites that appear between 25 and 35 ppm. Glu, glutamate; Tau, taurine; GABA, γ-aminobutyric acid; Gln, glutamine.
Figure 3.
 
The 13C NMR spectrum (low resolution) of metabolites from D2O extracts of the medium bathing dark-adapted ground squirrel retinas incubated for 2 hours in medium containing 13C-1 glucose.
Figure 3.
 
The 13C NMR spectrum (low resolution) of metabolites from D2O extracts of the medium bathing dark-adapted ground squirrel retinas incubated for 2 hours in medium containing 13C-1 glucose.
Figure 4.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of dark-adapted rat retinas incubated for 2 hours in medium containing 13C-1 glucose. Inset: metabolites that appear between 25 and 35 ppm. Glu, glutamate; Tau, taurine; GABA, γ-aminobutyric acid; Gln, glutamine.
Figure 4.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of dark-adapted rat retinas incubated for 2 hours in medium containing 13C-1 glucose. Inset: metabolites that appear between 25 and 35 ppm. Glu, glutamate; Tau, taurine; GABA, γ-aminobutyric acid; Gln, glutamine.
Figure 5.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of the medium bathing dark-adapted rat retinas incubated for 2 hours in medium containing 13C-1 glucose.
Figure 5.
 
The 13C NMR spectra (top: low resolution; bottom: high resolution) of metabolites from D2O extracts of the medium bathing dark-adapted rat retinas incubated for 2 hours in medium containing 13C-1 glucose.
Table 1.
 
Lactate Production in Ground Squirrel Retinas*
Table 1.
 
Lactate Production in Ground Squirrel Retinas*
Condition Inhibitors Aerobic Anaerobic Pasteur Effect
Dark None 4.5 ± 0.4 (9) 6.1 ± 0.3 (5) 1.4
Light None 3.7 ± 0.4 (8) 5.4 ± 0.3 (5) 1.5
Dark Cocktail 4.5 ± 0.5 (6)
Light Cocktail 3.5 ± 0.7 (5)
Dark Ouabain 3.3 ± 0.3 (8)
Light Ouabain 2.8 ± 0.5 (4)
Table 2.
 
14CO2 Production from 14C-3,4 Glucose in Ground Squirrel Retinas*
Table 2.
 
14CO2 Production from 14C-3,4 Glucose in Ground Squirrel Retinas*
Condition Inhibitors 14CO2
Dark None 0.11 ± 0.04 (5)
Light None 0.12 ± 0.05 (5)
Dark Cocktail 0.11 ± 0.04 (3)
Light Cocktail 0.08 ± 0.04 (3)
Dark Ouabain 0.04 ± 0.01 (4)
Light Ouabain 0.04 ± 0.01 (4)
Table 3.
 
Quantitative Estimates of Aerobic Glucose Utilization by Glycolysis and Mitochondrial Oxidation in Ground Squirrel Retinas*
Table 3.
 
Quantitative Estimates of Aerobic Glucose Utilization by Glycolysis and Mitochondrial Oxidation in Ground Squirrel Retinas*
Dark Light
Lactate Production 4.50 3.70
Glucose-to-lactate 2.25 1.85
Glucose oxidized by mitochondria 0.06 0.06
Glucose glycolyzed as a % of total utilized 96 94
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