Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1

Abstract

Tissues with high metabolic rates often use lipids, as well as glucose, for energy, conferring a survival advantage during feast and famine. Current dogma suggests that high-energy-consuming photoreceptors depend on glucose. Here we show that the retina also uses fatty acid β-oxidation for energy. Moreover, we identify a lipid sensor, free fatty acid receptor 1 (Ffar1), that curbs glucose uptake when fatty acids are available. Very-low-density lipoprotein receptor (Vldlr), which is present in photoreceptors and is expressed in other tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acid. In the retinas of Vldlr(-/-) mice with low fatty acid uptake but high circulating lipid levels, we found that Ffar1 suppresses expression of the glucose transporter Glut1. Impaired glucose entry into photoreceptors results in a dual (lipid and glucose) fuel shortage and a reduction in the levels of the Krebs cycle intermediate α-ketoglutarate (α-KG). Low α-KG levels promotes stabilization of hypoxia-induced factor 1a (Hif1a) and secretion of vascular endothelial growth factor A (Vegfa) by starved Vldlr(-/-) photoreceptors, leading to neovascularization. The aberrant vessels in the Vldlr(-/-) retinas, which invade normally avascular photoreceptors, are reminiscent of the vascular defects in retinal angiomatous proliferation, a subset of neovascular age-related macular degeneration (AMD), which is associated with high vitreous VEGFA levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in macular telangiectasia, neovascular AMD and other retinal diseases.

Figure 1. Retinal energy deficits are associated with vascular lesions in Vldlr^−/−

(a) Pathologic vessels in Vldlr^−/− retinas originated from the deep vascular plexus (DVP) and breached the outer plexiform layer (P12), extending towards photoreceptor outer segments (os) at P16. Scale: 200 µm. n = 5 retinas. (b) Vldlr^−/− pups raised in darkness (n = 10 retinas) compared to normal 12 hours light/dark cycle (Ctl: control, n = 28) to increase retinal energy demands. Scale: 1 mm (left), 0.5 mm (others). White spots label vascular lesions. P = 0.0031. (c) Mitochondrial volume quantified by 3D reconstruction of retinal scanning electron microscopy (SEM) images. Mitochondria within photoreceptors are pseudo-colored. n = 23 photoreceptors. Scale: 5µm. P < 0.0001. (d) Retinal ATP level was significantly lower in Vldlr^−/− retina (n = 6) compared to littermate control wild type retinas (WT; n = 4). P = 0.0026. Results are presented as mean ± SEM. Two-tailed Student t-test; ** P < 0.01, *** P < 0.001.

Figure 2. Dual lipid and glucose fuel deficiency in Vldlr^−/− retinas

(a) Oxygen consumption rate (OCR) and (b) maximal OCR of wild type (WT) retinas provided with long-chain fatty acid (FA) palmitate or control (Ctl: bovine serum albumin or BSA) in the presence or absence of FA oxidation inhibitor, etomoxir (40 µM). n = 6–8 retinas. (c) Circulating plasma palmitate levels in WT and Vldlr^−/− mice. n = 7 WT, 13 Vldlr^−/− mice plasma samples. P<0.0001. (d) Metabolite array of FA β-oxidation, (e) total acylcarnitine (P = 0.0108) and free carnitine levels (P = 0.0014) measured by LC/MS/MS. n = 3 animal retinas. (f) Cpt1a mRNA expression of intact retinas (left; P = 0.0052) and laser capture microdissected (LCM) retinal layers by qRT-PCR. ONL: outer nuclear (photoreceptors); INL: inner nuclear and GCL: ganglion cell layers. n = 3 animal retinas. (g) ¹⁸F-FDG microPET and CT scan revealed decreased glucose uptake in Vldlr^−/− retinas, confirmed by retinal gamma radioactivity counts. Scale: 4 mm. n = 22 WT, 12 Vldlr^−/− retinas, P = 0.0116. (h) Glut1 mRNA expression in intact retinas (left, n = 9 WT, 12 Vldlr^−/− retinas; P = 0.0119) and retinal layers (right) by LCM and qRT-PCR (n = 3 retinas). (i) Glut1 protein expression of intact WT and Vldlr^−/− retina. n = 6 retinas, P = 0.030. Results are presented as mean ± SEM. Two-tailed Student t-test (c–f, g–i) and one-way ANOVA with Tukey post-hoc analysis (b,f,h); * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 3. Ffar1 modulates retinal glucose uptake and RAP

(a) Decreased lipid uptake in Vldlr^−/− retina increased extracellular mid/long chain FA, the agonist of lipid sensor Ffar1, which was associated with reduced Glut1 expression. (b) Expression of FA sensing GPCR in WT and Vldlr^−/− intact retinas and (c) Ffar1 distribution in retinal layers by LCM (qRT-PCR). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. n = 3 animal retinas. (d) Glucose uptake (³H-2-DG tracer; n = 5–8 ctl, 9–16 GW-treated retinas), (e) Glut1 protein expression (n = 12 retinas, P < 0.0001) and (f) number of RAP-like pathologic vascular lesions at P16 in WT and Vldlr^−/− mice treated with Ffar1 agonist GW9508 (GW; n = 11) or vehicle (ctl; n = 7, P = 0.0002). (g) Glucose uptake (¹⁸F-FDG; n = 4 retinas, scale: 4 mm), (h) Glut1 protein expression (n = 10 WT, others 9 retinas), and (i) number of RAP-like pathologic vascular lesions of WT (no lesions) and Vldlr^−/− mice compared to littermate Vldlr^−/−/Ffar1^+/+ mice (P16; n = 10 retinas, P = 0.0153). Results are presented as mean ± SEM. Two-tailed Student t-test (e,f,i) and one-way ANOVA with Dunnett’s (b,c,g,h) or Tukey’s (d) post-hoc comparison; * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4. Fuel deficient Vldlr^−/− retina generates less α-Ketoglutarate and more Vegf

(a) Dual shortage of glucose (b, pyruvate; n = 15 WT, 12 Vldlr^−/− retinas P = 0.0032) and FA uptake reduces acetyl-coA (c, estimated by measuring acetylcarnitine; n = 3, P = 0.0094) and (d) TCA (Krebs) cycle intermediate α-KG in Vldlr^−/− retina (LC/MS/MS; n = 11 WT, 15 Vldlr^−/− retinas, P = 0.0069). Together with oxygen (O₂), α-KG is an essential co-activator of propyl-hydroxylase dehydrogenase (PHD) that tags HIF-1α for degradation by proline hydroxylation (hydroxyproline). (e) Levels of hydroxyproline residues in WT and Vldlr^−/− retinas measured by LC/MS/MS (n = 15 WT, 12 Vldlr^−/− animal retinas, P = 0.0004) and (f) Hif1a stabilization of WT, Vldlr^−/− and Vldlr^−/−/Ffar1^−/− retinal nuclear extractions. We used Fibrillarin (Fbl) as nuclear loading control. n = 3 all groups. (g) Hif1a retinal expression in Vldlr^−/− photoreceptor layer (P12 retinal flat mounts, Scale: 100 µm; left: extended focus; middle and right panels: 3D confocal IHC, n = 3) where (h) Vegfa was then also secreted (P16, ELISA, n = 6 retinas) and (i) localized (P16 retinal flat mounts, Scale: 100 µm; left: extended focus; middle and right panels: 3D confocal IHC, n = 3 retinas). (j) Human subjects with AMD, either retinal angiomatous proliferation (RAP, n = 3) or choroidal neovascularization (CNV, n = 7) had higher VEGFA vitreous levels by ELISA compared to control subjects without pathologic neovessels (macular hole; n = 8). Results are presented as mean ± SEM. Two-tailed Student t-test (c,d), Mann Whitney test (b,e), and one-way ANOVAs with post-hoc Dunett’s (f,j) or Tukey’s multiple comparison (h); * P < 0.05, ** P < 0.01, *** P < 0.001.