Hyperoxia induces glutamine-fuelled anaplerosis in retinal Müller cells

Singh, Charandeep; Tran, Vincent; McCollum, Leah; Bolok, Youstina; Allan, Kristin; Yuan, Alex; Hoppe, George; Brunengraber, Henri; Sears, Jonathan E.

doi:10.1038/s41467-020-15066-6

Cited by 21 publications

(18 citation statements)

References 41 publications

(44 reference statements)

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“…HIF-1α (#QT01039542) and VEGF (#QT00160769) were analyzed using primers from Qiagen. Primers for all other genes investigated are listed in the Gas Chromatograpy-Mass Spectrometry: As previously described (Singh et al, 2020), for each mouse, both retinas were dissected on ice cold HBSS and flash frozen. To each tube, 500 µl of -20°C 80% methanol was added with 20 µl of 0.05 mg/ml of [ 13 C5] ribitol as internal standard.…”

Section: Genotypingmentioning

confidence: 99%

Reduction of Glut1 in the Neural Retina But Not the RPE Alleviates Polyol Accumulation and Normalizes Early Characteristics of Diabetic Retinopathy

et al. 2021

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Hyperglycemia is a key determinant for development of diabetic retinopathy (DR). Inadequate glycemic control exacerbates retinopathy, while normalization of glucose levels delays its progression. In hyperglycemia, hexokinase is saturated and excess glucose is metabolized to sorbitol by aldose reductase via the polyol pathway. Therapies to reduce retinal polyol accumulation for the prevention of DR have been elusive due to low sorbitol dehydrogenase levels in the retina and inadequate inhibition of aldose reductase. Using systemic and conditional genetic inactivation, we targeted the primary facilitative glucose transporter in the retina, Glut1, as a preventative therapeutic in diabetic male and female mice.Unlike wildtype diabetics, diabetic Glut1 +/mice did not display elevated Glut1 levels in the retina. Furthermore, diabetic Glut1 +/mice exhibited ameliorated ERG defects, inflammation and oxidative stress, which was correlated with a significant reduction in retinal sorbitol accumulation. RPE-specific reduction of Glut1 did not prevent an increase in retinal sorbitol content or early hallmarks of DR. However, like diabetic Glut1 +/mice, reduction of Glut1 specifically in retinal neurons mitigated polyol accumulation and completely prevented retinal dysfunction and the elevation of markers for oxidative stress and inflammation associated with diabetes. These results suggest that modulation of retinal polyol accumulation via Glut1 in photoreceptors can circumvent the difficulties in regulating systemic glucose metabolism and be exploited to prevent DR.Significance: Diabetic retinopathy (DR) affects one third of diabetic patients and is the primary cause of vision loss in adults aged 20-74. While anti-VEGF and photocoagulation treatments for the late-stage vision threatening complications can prevent vision loss, a significant proportion of patients do not respond to anti-VEGF therapies and mechanisms to stop progression of early-stage symptoms remain elusive. Glut1 is the primary facilitative glucose transporter for the retina. We determined that a moderate reduction in Glut1 levels, specifically in retinal neurons, but not the RPE, was sufficient to prevent retinal

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Section: Genotypingmentioning

confidence: 99%

Reduction of Glut1 in the Neural Retina But Not the RPE Alleviates Polyol Accumulation and Normalizes Early Characteristics of Diabetic Retinopathy

et al. 2021

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“…Biomass synthesis pathways like serine/one-carbon and glutaminolysis involve Myc protein. These pathways were found to be altered by hyperoxia in our previous studies (Singh et al, 2019;Singh et al, 2018;Singh et al, 2020).…”

Section: Discussionmentioning

confidence: 70%

“…Six-hundred microliters of supernatant was transferred to fresh tubes and dried under vacuum in a -4°C cold vacuum evaporator (Labconco). Samples were derivatized with the two step protocol as described earlier (Singh et. al.…”

Section: Metabolite Extractionmentioning

confidence: 99%

Hyperoxia inhibits proliferation of retinal endothelial cells in Myc dependent manner

Singh

Benos

Grenell

et al. 2020

Preprint

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Oxygen supplementation is necessary to prevent mortality of severely premature infants. However, the supraphysiological concentration of oxygen utilized in these infants simultaneously creates retinovascular growth attenuation and vasoobliteration that induces retinopathy of prematurity. Here, we report that hyperoxia regulates the cell cycle and retinal endothelial cell proliferation in a previously unknown Myc dependent manner which contributes to oxygen-induced retinopathy.

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“…We have evaluated the strategy of stabilizing HIF during hyperoxia using protection against vascular obliteration and pathological angiogenesis measured in retinal flatmounts as one metric of efficacy. In retinas protected against retinopathy we find (1) no liver or kidney toxicity, (2) 85-100% reduction of oxygen induced retinal vasoobliteration and neovascularization in mice and rats, (3) normalization of the electroretinogram in hyperoxia, (4) normalization of ocular coherence tomography, (5) a cadre of serum biomarkers secreted by liver, and (6) and a metabolic switch to upregulate the urea cycle and 1C/serine metabolism (Hoppe et al, 2014(Hoppe et al, , 2016Singh et al, 2019Singh et al, , 2020. We observed that when using DMOG, hepatic HIF-1 was necessary and sufficient to prevent OIR but that Roxadustat could partially overcome hepatic HIF-1 ablation because it also targeted the retina.…”

Section: Introductionmentioning

confidence: 87%

Rank Order of Small Molecule Induced Hypoxiamimesis to Prevent Retinopathy of Prematurity

Hoppe

Bolok

McCollum

et al. 2020

Front. Cell Dev. Biol.

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Here we rank order small molecule inhibitors of hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) using severity of oxygen induced retinopathy (OIR) as an outcome measure. Dose response analyses in cell cultures of hepatoma (Hep3B), retinal Müller cells (MIO-M1) and primary retinal endothelial cells were conducted to evaluate potency by comparing dose to HIF-1,2 protein levels by western blotting. In vivo dose response was determined using the luciferase-transgene HIF reporter (luc-ODD). Each compound was placed in rank order by their ability to reduce neovascularization and capillary drop out in the OIR mouse model. An Epas1 KO confined to retinal Müller cells was used to determine whether successful protection by HIF stabilization requires HIF-2. Two candidate small molecules can prevent OIR by stabilizing HIF-1 to prevent oxygen induced growth attenuation and vascular obliteration. Müller cell HIF-2, the mediator of pathologic retinal angiogenesis, is not required for protection. The lack of dependence on Müller cell HIF-2 predicts that inhibition of HIF PHD will not drive pathological angiogenesis.

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Hyperoxia induces glutamine-fuelled anaplerosis in retinal Müller cells

Cited by 21 publications

References 41 publications

Reduction of Glut1 in the Neural Retina But Not the RPE Alleviates Polyol Accumulation and Normalizes Early Characteristics of Diabetic Retinopathy

Reduction of Glut1 in the Neural Retina But Not the RPE Alleviates Polyol Accumulation and Normalizes Early Characteristics of Diabetic Retinopathy

Hyperoxia inhibits proliferation of retinal endothelial cells in Myc dependent manner

Rank Order of Small Molecule Induced Hypoxiamimesis to Prevent Retinopathy of Prematurity

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