Glucose uptake by GLUT1 in photoreceptors is essential for outer segment renewal and rod photoreceptor survival

Abstract

Photoreceptors consume glucose supplied by the choriocapillaris to support phototransduction and outer segment (OS) renewal. Reduced glucose supply underlies photoreceptor cell death in inherited retinal degeneration and age-related retinal disease. We have previously shown that restricting glucose transport into the outer retina by conditional deletion of Slc2a1 encoding GLUT1 resulted in photoreceptor loss and impaired OS renewal. However, retinal neurons, glia, and the retinal pigment epithelium play specialized, synergistic roles in metabolite supply and exchange, and the cell-specific map of glucose uptake and utilization in the retina is incomplete. In these studies, we conditionally deleted Slc2a1 in a pan-retinal or rod-specific manner to better understand how glucose is utilized in the retina. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Slc2a1 from retinal neurons and Müller glia results in reduced OS growth and progressive rod but not cone photoreceptor cell death. Rhodopsin levels were severely decreased even at postnatal day 20 when OS length was relatively normal. Arrestin levels were not changed suggesting that glucose uptake is required to synthesize membrane glycoproteins. Rod-specific deletion of Slc2a1 resulted in similar changes in OS length and rod photoreceptor cell death. These studies demonstrate that glucose is an essential carbon source for rod photoreceptor cell OS maintenance and viability.

Keywords: Slc2a1; GLUT1; glucose depervation; photoreceptors; retina; rhodopsin.

FIGURE 1

GLUT1 is the primary glucose transporter in the outer retina (A) scRNAseq analysis of genes encoding solute transporters with glucose transport activity (GO:5355) from the 1‐month‐old murine retina (GSE153674). Each cell type shows the Log 2 transformed average transcript per million (TPM). (B) Expression of Slc2a1 and Slc2a3 in control mouse retinas using in situ hybridization. The left panel shows Slc2a1 transcript, the right panel shows Slc2a3 transcript detected (red) (Scale bar indicates 50 μm).

FIGURE 2

Deletion of GLUT1 from the retina does not result in compensatory changes in expression of GLUT3 (A) In situ hybridization to detect the localization of Slc2a1. The Ret∆Glut1 mice had a loss of Slc2a1 (red) signal in the inner and outer retina with intermittent expression. Slc2a1 expression is seen in inner retinal blood vessels (as indicated by white arrows) and the RPE (Scale bar indicates 50 μm). (B) Immunostaining of GLUT1 (green) in control and Ret∆Glut1 mice. White arrows indicate GLUT1 expression in inner retinal blood vessels. (Scale bar indicates 50 μm). (C) In situ hybridization of Slc2a3 from control and RetΔGlut1 mice at 1 month of age. (Scale bar indicates 50 μm). (D) Immunostaining of GLUT3 (green) in control and Ret∆Glut1 mice at 1 month of age. (Scale bar indicates 50 μm). (E) Western blot analysis of GLUT1 expression in control and Ret∆Glut1 retinas. 5 μg retina protein was loaded per well. Blots are representative of N = 5. Bars indicate the average ± SD of GLUT1 intensity normalized to β‐Actin for N = 5 mice. (F) Western blot analysis of GLUT3 expression in control and Ret∆Glut1 retinas. 5 μg retina protein was loaded per well. Blots are representative of N = 3. Bars indicate the average ± SD of GLUT3 intensity normalized to Vinculin for N = 3 mice.

FIGURE 3

Rods degenerate in mice lacking retinal GLUT1 expression (A) Averaged SD‐OCT B‐scan from the horizontal meridian of retinas from 2‐month‐old control and 1, 2, and 4‐month‐old RetΔGlut1 mice. GCL, INL, OPL, ONL, IS, OS, and RPE are indicated. (B) ONL layer thickness was measured from volumetric SD‐OCT scans at P20, 1, 2, and 4 months of age. Data points indicate average (± SD) for 3–6 mice. (C) Inner Retinal Thickness (OPL‐GCL) was measured from volumetric SD‐OCT scans at P20, 1, 2, and 4 months of age. Data points indicate average (± SD) for 3–6 mice. (D) Luminance‐response functions for a‐waves and b‐waves were recorded from 1‐month mice. Data points indicate average (± SEM) for 13–14 mice. (E) Maximum amplitudes for a‐waves (at 1.4 log cd · s/m² luminance) at the ages indicated. Bars indicate the average (± SEM) for 8–14 mice. (F) OS length was measured from volumetric SD‐OCT scans at P20, 1, 2, and 4 months of age. Data points indicate average (± SD) for 3–6 mice.

FIGURE 4

Cones of RetΔGlut1 mice have impaired function but do not degenerate (A) Fluorescence images of retinal flat mounts immunolabeled for cone arrestin ARR3 (red). The scale bar indicates 100 μm. Box plots of estimations of cone density per mm² at various radial positions halfway between optic nerve and edge of the retina in flattened retinas for 3 mice. (B) Luminance response functions from photopic ERG b‐waves were recorded from 1‐month control and RetΔGlut1 mice under light‐adapted conditions. Data points indicate average (± SEM) for 13–14 mice. (C) Maximum amplitudes for photopic b‐waves (for flashes at 1.4 log cd · s/m² luminance) for mice at the ages indicated. Bars indicate an average (± SEM) for 8–14 mice.

FIGURE 5

Reduced opsin synthesis contributes to the impaired renewal of rod and cone OS (A) Left: Retina cryosections from control and RetΔGlut1 mice immunolabeled for RHO (red) and counterstained with DAPI (blue) at P12 (top row) and P30 (bottom row). OS lengths were estimated with ImageJ from images of cryosections like those shown from P12 and 1‐month control and RetΔGlut1 mice. Right: Bars indicate the average ± SD for 3 mice. Scale bar indicates 50 μm. (B) Left: Retina cryosections from control and RetΔGlut1 mice immunolabeled for cone opsin (green) and counterstained with DAPI (blue) at P12 (top row) and P30 (bottom row). OS lengths were estimated with ImageJ from images like those shown at left, from P12 and 1‐month control and RetΔGlut1 mice. Right: Bars indicate the average ± SD for 3 mice. The scale bar indicates 50 μm. (C) Western blots showing relative GLUT1 (5 μg/well), Rhodopsin (RHO) (0.25 μg/well), and rod arrestin (ARR1) (0.25 μg/well) levels in retina lysates from control and RetΔGlut1 mice aged P12, P20, and P30. The ratio of density signals for rhodopsin relative to ARR1 at various ages was estimated from the retinas of control and RetΔGlut1 mice. Data points indicate average ± SD for 3 mice. (D) Relative transcription levels for rhodopsin (Rho) and rod arrestin (Arr1) estimated from real‐time RT‐PCR analysis with probes for mouse, rho, and Arr1 for retinas of P30 control and RetΔGlut1 mice. Bars indicate the average ± SD for 3 mice. (E) Left: Western blots showing relative M‐opsin and cone arrestin (ARR3) levels in retina lysates (2.5 μg/well) from control and RetΔGlut1 mice aged P30. Right: Ratio of density signals for M‐opsin and ARR3 relative to vinculin at P30 estimated from retinas of control and RetΔGlut1 mice. Bars indicate the average ± SD for 3 mice. (F) Relative transcription levels for M‐opsin and cone arrestin estimated from real‐time RT‐PCR analysis with probes for mouse Opn1mw and (Arr3) for retinas of P30 control and RetΔGlut1 mice. Bars indicate the average ± SD for 3 mice.

FIGURE 6

Increased inflammation in the outer retina of RetΔGlut1 mice (A) Representative 55° wide‐field BAF‐cSLO images obtained from 2‐month control and 1‐, 2‐, and 4‐month RetΔGlut1 mice. (B) The number of BAF‐cSLO identified hyperfluorescent foci was quantified at each age in RetΔGlut1 and control mice. Data points indicate average ± SD for 3–6 mice. (C) Iba‐1 immunofluorescence (magenta) in retina cryosections from control, 2 and 4 months RetΔGlut1 mice counterstained with Phalloidin (green) DAPI (gray). Scale bar indicates 50 μm. IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer, INL, inner nuclear layer; IPL, inner plexiform layer, GCL, ganglion cell layer (D) Left: TUNEL labeling (green) of retina cryosections from P15 control and RetΔGlut1 mice, counterstained with DAPI (gray). The scale bar indicates 50 μm. Right: quantification of TUNEL per 0.2 mm length retina from immunofluorescent images. Bars indicate the average ± SD for 2 mice. (E) Left: Avidin labeling (green) of retina cryosections from control and RetΔGlut1 at P15 with DAPI counterstain (gray). The scale bar indicates 25 μm. Right: Quantification of avidin positive cells in oriented fluorescence images within 0.2 mm wide field of view. Bars indicate the average ± SD for 3 mice. GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segments; ONL, outer nuclear layer; RPE, retinal pigmented epithelium.

FIGURE 7

RetΔGlut1 mice have reduced lactate efflux and an altered metabolic profile (A) Lactate efflux from isolated retinas incubated for 1 h in Ringer's with 5 mM glucose from control and RetΔGlut1 mice. Bars indicate the average ± SEM for 4 retinas. (B) Partial Least‐Squares Discriminant Analysis (PLS‐DA) of Control and Ret∆Glut1 samples from LC–MS data. Data points indicate results from 5 control and 3 RetΔGlut1. (C) Metabolomics analysis of significant metabolites changed in Ret∆Glut1 retina samples. Raw data were normalized to the control average (hashed line), and statistical significance was obtained from T‐tests after Pareto scaling. Bars indicate the average (± SD) for 3–5 mice (D) Heat map of top 25 metabolites changed based on the statistical significance or fold change between control and Ret∆Glut1 retina samples, after Pareto scaling.

FIGURE 8

Loss of GLUT1 from rods results in impaired OS turnover and reduced ERG amplitude, but impacts are less severe than in Ret∆Glut1 (A) In situ hybridization for Slc2a1 transcripts (red) in control and Rod∆Glut1. The scale bar indicates 50 μm. (B) Immunofluorescence for GLUT1 (green) in control and Rod∆Glut1 retinas. The scale bar indicates 50 μm. (C) Western blot analysis of GLUT1 expression in control, Ret∆Glut1, and Rod∆Glut1 retinas (5 μg/well). Blots are representative of N = 3 mice. Quantification of relative expression of GLUT1 in retinas of control, Ret∆Glut1, and Rod∆Glut1. The intensity of GLUT1 was normalized to Vinculin. Bars indicate the average (± SD) for 3 mice. (D) Retina cryosections from control and RodΔGlut1 mice 1‐month post‐tamoxifen injection immunolabeled for RHO (red) and counterstained with DAPI (blue). The scale bar indicates 25 μm. ONL thickness and OS lengths were estimated with ImageJ. Bars indicate the average (± SD) for 3 mice. (E) Luminance response plots from peak amplitudes of scotopic a‐waves and b‐waves were recorded from control and RodΔGlut1 mice 1‐month post‐tamoxifen injection. Data points indicate average (± SEM) for 7 mice (F) Luminance response plots from photopic b‐waves of control and RodΔGlut1 mice 1‐month post‐tamoxifen injection. Data points indicate average (± SEM) for 7 mice (G) Maximum amplitudes for scotopic a‐ and b‐ waves (S:a‐wave, and S:b‐wave) and photopic b‐wave (P:b‐wave) at 1.4 log cd · s/m² luminance. Bars indicate the average (± SD) for 7 mice. (H) Left: Western blot analysis of expression of RHO (0.25 μg/well), and ARR1 (0.25 μg/well) in control and RodΔGlut1 mice 1‐month post‐tamoxifen injection. Right: Bars indicate the average (± SD) for 5–7 mice.