Biology subjects

Sadilek, M.

Publications and source records attributed to Sadilek, M..

6 recordsLinked to original sources

bioRxiv2023

Acetyl-CoA carboxylase inhibition increases RPE cell fatty acid oxidation and limits apolipoprotein efflux

PurposeIn age-related macular degeneration (AMD) and Sorsbys fundus dystrophy (SFD), lipid-rich deposits known as drusen accumulate under the retinal pigment epithelium (RPE). Drusen may contribute to photoreceptor and RPE degeneration in AMD and SFD. We hypothesize that stimulating {beta}-oxidation in RPE will reduce drusen accumulation. Inhibitors of acetyl-CoA carboxylase (ACC) stimulate {beta}-oxidation and diminish lipid accumulation in fatty liver disease. In this report we test the hypothesis that an ACC inhibitor, Firsocostat, limits the accumulation of lipid deposits in cultured RPE cells. MethodsWe probed metabolism and cellular function in mouse RPE-choroid, human fetal- derived RPE cells, and induced pluripotent stem cell-derived RPE cells. We used 13C6-glucose and 13C16-palmitate to determine the effects of Firsocostat on glycolytic, Krebs cycle, and fatty acid metabolism. 13C labeling of metabolites in these pathways were analyzed using gas chromatography-linked mass spectrometry. We quantified ApoE and VEGF release using enzyme-linked immunosorbent assays. Immunostaining of sectioned RPE was used to visualize ApoE deposits. RPE function was assessed by measuring the trans-epithelial electrical resistance (TEER). ResultsACC inhibition with Firsocostat increases fatty acid oxidation and remodels lipid composition, glycolytic metabolism, lipoprotein release, and enhances TEER. When human serum is used to induce sub-RPE lipoprotein accumulation, fewer lipoproteins accumulate with Firsocostat. In a culture model of Sorsbys fundus dystrophy, Firsocostat also stimulates fatty acid oxidation, improves morphology, and increases TEER. ConclusionsFirsocostat remodels intracellular metabolism and improves RPE resilience to serum-induced lipid deposition. This effect of ACC inhibition suggests that it could be an effective strategy for diminishing drusen accumulation in the eyes of patients with AMD.

biochemistry↗

bioRxiv2021

Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

Post-translational modification of histone H4 by the small ubiquitin-like modifier (SUMO) protein was associated with gene repression. However, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using H4 chemically modified at Lys12 by SUMO-3 (H4K12su) that was incorporated into mononucleosomes and chromatinized plasmids. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, and transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed H4K12su inhibits RNA polymerase II-mediated transcription and H4 tail acetylation. Thus, we have uncovered negative biochemical crosstalk with acetylation/methylation and the direct inhibition of RNAPII-mediated transcription by H4K12su.

biochemistry↗

bioRxiv2021

Metabolic function in aging retina and retinal pigment epithelium remains robust despite vision loss

PurposeCharacterize how metabolic function in the murine retina and retinal pigment epithelium-choroid-sclera (eyecup) complex is impacted by natural aging. MethodsWe examined scotopic and photopic visual function of young (3-6 months) and aged (23-26 months) C57Bl/6J mice using electroretinograms (ERGs). Metabolic changes in retina and eyecup explants were characterized by measuring uptake and usage of U-13C-glucose or U-13C-glutamine at different timepoints by gas chromatography-mass spectrometry (GC-MS), measuring oxygen consumption rate (OCR) using a perifusion apparatus, and determining ATP levels with a bioluminescence assay. ResultsScotopic and photopic ERG responses declined in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected by the age of the mouse. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. ConclusionsThe ex vivo approach in our study to examine aging glucose and glutamine metabolism in retina and RPE showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The surprising metabolic stability of these tissues ex vivo suggests age-related metabolic alterations in these tissues may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or other structural changes influence ocular metabolism in vivo.

biochemistry↗

bioRxiv2021

Succinate is Broadly Tissue-Permeant and Uncouples Mitochondrial Respiration from ATP Synthesis

Succinate is central in energy metabolism. It is thought to be made and consumed within mitochondria, and not to cross the plasma membrane. Contrary to the canonical role of succinate, it is produced in the retina and released into the extracellular milleau. It is unknown how this affects adjacent retinal pigment epithelium, within eyecup tissue. By measuring consumption of 13C4-succinate and O2 we probe metabolism of the eyecup, we find robust succinate consumption by eyecup but not retina tissue. Succinate-dependant stimulation of eyecup respiration is oligomycin resistant; indicating that in eyecup tissue succinate uncouples mitochondrial ATP synthesis from respiration.

biochemistry↗

bioRxiv2020

Succinate can shuttle reducing power from the hypoxic retina to the O2-rich pigment epithelium.

When O2 is plentiful, the mitochondrial electron transport chain uses it as a terminal electron acceptor. However, the mammalian retina thrives within a hypoxic niche in the eye. We find that mitochondria in retinas adapt to their hypoxic environment by relying on the "reverse" succinate dehydrogenase reaction, where fumarate accepts electrons instead of O2. Reverse succinate dehydrogenase activity produces succinate and is enhanced by down-regulation of cytochrome oxidase subunits. Retinas can export the succinate to the neighboring retinal pigment epithelium/choroid complex. There, succinate stimulates O2 consumption several fold and enhances synthesis and release of malate. Malate released from the pigment epithelium can be imported into the retina, where it is converted to fumarate and again used to accept electrons in the reverse succinate dehydrogenase reaction. Our findings show how a malate/succinate shuttle can sustain these two tissues by transferring reducing power from an O2-poor tissue (retina) to an O2-rich one (retinal pigment epithelium/choroid).

biochemistry↗

bioRxiv2017

Biochemical Adaptations Of The Retina And Retinal Pigment Epithelium Support A Metabolic Ecosystem In The Vertebrate Eye

Here we report multiple lines of evidence for a comprehensive model for retinal energy metabolism. Metabolic flux, locations of key enzymes and our finding that glucose enters the neural retina almost entirely through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood supply passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Muller glial cells. A key feature of this model is that aerobic glycolysis in photoreceptors produces lactate to suppress glycolysis in the neighboring retinal pigment epithelium. That enhances the flow of glucose to the retina by minimizing consumption of glucose within the retinal pigment epithelium. This framework for metabolic relationships in retina provides new insights into the underlying causes of retinal disease, age-related vision loss and metabolism-based therapies.

biochemistry↗