Biology subjects

Philp, N. J.

Publications and source records attributed to Philp, N. J..

4 recordsLinked to original sources

bioRxiv2024

Reassessing Retinal Pigment Epithelial Ketogenesis: Enzymatic Assays for Ketone Body Levels Provide Inaccurate Results

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate ({beta}-HB). Prior work, based on detecting {beta}-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export {beta}-HB across the apical membrane. Here, we compare the accuracy of measuring {beta}-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of {beta}-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete {beta}-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming {beta}-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce {beta}-HB. Altogether, we substantiate {beta}-HB secretion in RPE but find that the secretion is equal apically and basally, RPE {beta}-HB can derive from ketogenic amino acids or fatty acids, and accurate {beta}-HB assessment requires mass spectrometric analysis.

cell biology↗

bioRxiv2024

Lactate transport inhibition therapeutically reprograms fibroblast metabolism in experimental pulmonary fibrosis

Myofibroblast differentiation, essential for driving extracellular matrix synthesis in pulmonary fibrosis, requires increased glycolysis. While glycolytic cells must export lactate, the contributions of lactate transporters to myofibroblast differentiation are unknown. In this study, we investigated how MCT1 and MCT4, key lactate transporters, influence myofibroblast differentiation and experimental pulmonary fibrosis. Our findings reveal that inhibiting MCT1 or MCT4 reduces TGF{beta}-stimulated pulmonary myofibroblast differentiation in vitro and decreases bleomycin-induced pulmonary fibrosis in vivo. Through comprehensive metabolic analyses, including bioenergetics, stable isotope tracing, metabolomics, and imaging mass spectrometry in both cells and mice, we demonstrate that inhibiting lactate transport enhances oxidative phosphorylation, reduces reactive oxygen species production, and diminishes glucose metabolite incorporation into fibrotic lung regions. Furthermore, we introduce VB253, a novel MCT4 inhibitor, which ameliorates pulmonary fibrosis in both young and aged mice, with comparable efficacy to established antifibrotic therapies. These results underscore the necessity of lactate transport for myofibroblast differentiation, identify MCT1 and MCT4 as promising pharmacologic targets in pulmonary fibrosis, and support further evaluation of lactate transport inhibitors for patients for whom limited therapeutic options currently exist. SUMMARYSmall molecule inhibitors of lactate transporters, including the novel MCT4 inhibitor VB253, reprogram fibroblast metabolism to prevent myofibroblast differentiation and decrease bleomycin-induced pulmonary fibrosis.

cell biology↗

bioRxiv2023

Glucose Hypometabolism Prompts RAN Translation and Exacerbates C9orf72-related ALS/FTD Phenotypes

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, although its potential role in disease pathogenesis is unknown. Here, we identified alterations in glucose metabolic pathways and ATP levels in the brain of asymptomatic C9-BAC mice. We found that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also found that one of the arginine-rich DPRs (PR) can directly contribute to glucose metabolism and metabolic stress. These findings provide a mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and support a feedforward loop model that opens several opportunities for therapeutic intervention.

neuroscience↗

bioRxiv2019

Peroxisome turnover and diurnal modulation of antioxidant activity in retinal pigment epithelia utilizes microtubule-associated protein 1 light chain 3B

The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid the visual cycle, nutrient supply, ion exchange and waste removal. Each day the RPE removes the oldest [~]10% of photoreceptor outer segments through phagocytic uptake, which peaks in a synchronous burst following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation and cell death. OSs are rich in very long chain fatty acids which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblotting and catalase activity assays, we investigated peroxisome abundance and activity at 6 am, 7 am (at lights on), 8 am, and 3 pm, in WT mice and mice lacking microtubule-associated protein 1 light chain 3B (LC3B), that have impaired degradation of phagosomes. We found that catalase activity, but not protein expression, is 50% higher in the morning compared with 3 pm, in RPE of WT but not LC3B-/- mice. Surprisingly, we found that peroxisome abundance was stable during the day, however numbers are elevated overall in LC3B-/- mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.

biochemistry↗