Glucose is the primary fuel for retina metabolism.

Jugular vein and carotid artery catheters were installed in C57BL/6J mice. After mice had recovered from the surgery, they were infused with 13C6-glucose, 13C3-lactate, 13C5-glutamine, 13C4-succinate, and 13C4-malate. Infusion parameters are listed in Table 1. Shown in the figure are schematics of (A) the setup for infusion experiments, and (B) the pathways through which infused fuels (highlighted by yellow ovals) integrate into energy metabolism. Each circle represents a carbon atom on an intermediate in these pathways. Blue circles represent labeling patterns from glucose or lactate, while purple circles represent labeling patterns from glutamine, succinate, and malate. After mice are infused with each tracer, they are euthanized and their retinas dissected then. Tissue and plasma samples are analyzed by mass spectrometry. C-F show the labeling of (C) pyruvate, (D) citrate, (E) glutamate, and (F) aspartate from infused 13C6-glucose (n=4), 13C3- lactate (n=7), 13C5-glutamine (n=4), 13C4-succinate (n=4) or 13C4-malate (n=4). “Normalized labeling” indicates the labeling of the metabolic intermediate in the figure panel divided by the labeling of the infused intermediate in circulation. This normalization method accounts for differences in the proportion of circulating fuel in the bloodstream.

Stock concentrations and infusion rates for 13C-labeled fuels.

Photoreceptors are the main consumers of glucose in the retina.

Retinas were dissected from eyes then incubated in Krebs-Ringer Bicarbonate buffer supplemented with 5 mM glucose, at 37°C and 5% CO2. Glucose consumption from medium (A, D, F) and lactate production (B, E, G) were monitored over 1-1.5 hours. These assays were used to determine how glycolytic metabolism is affected by (A, B) photoreceptor degeneration due to loss of AIPL1 (n=3-4), (D, E) loss of Glut1 from the retina (n=4-5), or (F, G) loss of Glut1 from rod photoreceptors (n=5). Loss of photoreceptors, loss of retinal Glut1, or loss of rod Glut1 slowed retina glucose utilization. (C) C57BL/6J and AIPL1-/- retinas were also incubated in KRB supplemented with 5 mM 13C6-glucose. Retina tissue was flash-frozen 0, 0.5, 1, 3, or 5 minutes after the start of the incubation. Labeling of metabolic intermediates in the retina was assessed by mass spectrometry. Labeling of lactate with 13C from glucose is slowed by the loss of photoreceptors (n=3-4 retinas / time point).

When retinas cannot consume glucose, they consume more lactate.

Mice lacking Glut1 in rod photoreceptors (RodΔGlut1) and identically treated littermate control mice were euthanized, and retinas dissected. Retinas were incubated for 2 hours in KRB supplemented with 5 mM glucose, 10 mM lactate, or 100 µM palmitate-BSA. In this experiment, source of 13C was 13C6-glucose for one cohort of retinas, 13C3-lactate for the second cohort, or 13C16-palmitate in a third cohort. Following the incubation, retinas were flash-frozen in liquid N2 and later analyzed by mass spectrometry. Our hypothesis is that loss of glucose metabolism in rods would increase metabolism from the other fuel sources by altering NAD+/NADH. This hypothesis is illustrated in (A). (B) in control retinas, the level of labeling on PEP, alanine, pyruvate, citrate, glutamine, malate, or aspartate from 13C6-glucose, 13C3-lactate, or 13C16-palmitate. Glucose and lactate are the most prevalent sources of 13C. Fractional labeling of glycolytic, TCA cycle intermediates, and amino acids in control and RodΔGlut1 retinas from (C) 13C6-glucose (n=3-4), (D) 13C3-lactate (n=3-4), or (E) 13C16-palmitate (n=4). Data are displayed as a fold-change from labeling in control retinas.

When photoreceptors do not consume glucose, there is a decrease in RPE- choroid lactate.

Aipl1-/-, Pde6bH620Q, RetΔGlut1, RodΔGlut1, and control mice were euthanized and RPE-choroid tissue was immediately dissected from the eye and flash-frozen. Tissue metabolite levels were analyzed using mass spectrometry, and divided into three categories; glycolytic intermediates (A, D, G, J), TCA cycle intermediates (B, E, H, K), and amino acids (C, F, I, L). Metabolite abundances in Aipl1-/- (A-C) and Pde6bH620QRPE-choroid tissue (D-F) was compared to metabolite abundances in RPE-choroid tissue from C57BL/6J controls of a similar age. Metabolite abundances in RetΔGlut1 (G-I) and RodΔGlut1 RPE- choroid tissue (J-L) was compared to metabolite abundances in RPE-choroid tissue from uninjected or tamoxifen injected Glut1fl/fl controls of a similar age. Lactate levels were consistently lower in the experimental groups of all tissues. n=5-6 per group.

Mouse RPE-choroid uses photoreceptor-derived lactate in vivo.

(A, B) C57BL/6J and Aipl1 -/- mice were euthanized and their (A) retina or (B) RPE-choroid tissue were incubated ex vivo in 13C6-glucose. The amount of 13C labeled DHAP, PEP, pyruvate, lactate, citrate, succinate, and malate were quantified by mass spectrometry. Aipl1-/- decreases utilization of glucose by the retina, but not the RPE-choroid. (C, D) Jugular vein catheters were installed in C57BL/6J and Aipl1-/- mice. A bolus dose of 100 mg/kg 13C6-glucose was infused through jugular vein catheters, then mice were euthanized 1, 2, 3, or 5 minutes later. Tissue levels and 13C labeling of DHAP, PEP, pyruvate, lactate, citrate, succinate, and malate were determined using mass spectrometry. 13C labeling of labeling of metabolic intermediates was drastically altered in both the (C) retina and (D) RPE-choroid of mice where the 13C6-glucose had been infused. (E, F) contain data from the same mice as C and D, but show the proportion of each metabolite that is 13C labeled. In all panels, “0 minute” samples were from retina or RPE-choroid tissue that was not exposed to a 13C fuel.