Serine and 1-carbon metabolism are required for HIF-mediated protection against retinopathy of prematurity
Charandeep Singh, George Hoppe, Vincent Tran, Leah McCollum, Youstina Bolok, Weilin Song, Amit Sharma, Henri Brunengraber, Jonathan E. Sears
Charandeep Singh, George Hoppe, Vincent Tran, Leah McCollum, Youstina Bolok, Weilin Song, Amit Sharma, Henri Brunengraber, Jonathan E. Sears
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Research Article Angiogenesis Metabolism

Serine and 1-carbon metabolism are required for HIF-mediated protection against retinopathy of prematurity

Abstract

We determined which metabolic pathways are activated by hypoxia-inducible factor 1–mediated (HIF-1–mediated) protection against oxygen-induced retinopathy (OIR) in newborn mice, the experimental correlate to retinopathy of prematurity, a leading cause of infant blindness. HIF-1 coordinates the change from oxidative to glycolytic metabolism and mediates flux through serine and 1-carbon metabolism (1CM) in hypoxic and cancer cells. We used untargeted metabolite profiling in vivo to demonstrate that hypoxia mimesis activates serine/1CM. Both [13C6] glucose labeling of metabolites in ex vivo retinal explants as well as in vivo [13C3] serine labeling of metabolites followed in liver lysates strongly suggest that retinal serine is primarily derived from hepatic glycolytic carbon and not from retinal glycolytic carbon in newborn pups. In HIF-1α2lox/2lox albumin-Cre–knockout mice, reduced or near-0 levels of serine/glycine further demonstrate the hepatic origin of retinal serine. Furthermore, inhibition of 1CM by methotrexate blocked HIF-mediated protection against OIR. This demonstrated that 1CM participates in protection induced by HIF-1 stabilization. The urea cycle also dominated pathway enrichment analyses of plasma samples. The dependence of retinal serine on hepatic HIF-1 and the upregulation of the urea cycle emphasize the importance of the liver to remote protection of the retina.

Authors

Charandeep Singh, George Hoppe, Vincent Tran, Leah McCollum, Youstina Bolok, Weilin Song, Amit Sharma, Henri Brunengraber, Jonathan E. Sears

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Figure 3

Hepatic 1CM drives protection against OIR.

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Hepatic 1CM drives protection against OIR. (A) Schematic showing atom tr...
(A) Schematic showing atom transitions in all aspects of 1CM. (B) Quantification of newborn mice treated with PBS or PBS plus methotrexate (MTX) compared with Roxadustat (RXD) or RXD plus MTX. There was increased vaso-obliteration in the RXD plus MTX group. MTX did not worsen the control group but did worsen the RXD group, showing that hyperoxia downregulates 1CM to such an extent that it is not additive to creating pathology. Under HIF stabilization, 1CM is upregulated and this is blocked by MTX. (C) Quantification of pathological angiogenesis or neovascularization shows that 1CM has no effect on proliferation presumably because it was administered only in phase 1. (D) Representative flat mount from control PBS-injected newborn mouse shows capillary dropout (yellow) and neovascularization (red). (E) Mouse pup retinal flat mount protected by HIF stabilization using RXD. (F) MTX plus RXD (RXD + MTX) demonstrates reduction in protection when 1CM is blocked by dihydrofolate reductase. (G) [13C3] serine injected subcutaneously was followed in liver extracts, demonstrating conversion to M2 glycine, M2 hypotaurine, and M2 taurine, all metabolites of 1CM. (H) The change in retinal serine and glycine using a hepatic-specific HIF stabilizer in wild-type (WT) versus HIF-1α2lox/2lox albumin-Cre–knockout (HIF-1α–KO) mice. (n = 6 animals in each group.) (I) Serum serine and glycine levels in the WT versus HIF-1α2lox/2lox, DMOG-injected mouse metabolite extracts. Metabolites were extracted from the serum at different time points after DMOG i.p. injections. *, 2-tailed t test P values less than 0.05.