Methionine as a regulator of bone remodeling with fasting.

Caloric restriction improves metabolic health but is often complicated by bone loss. We studied bone parameters in humans during a 10-day fast and identified candidate metabolic regulators of bone turnover. Pro-collagen 1 intact N-terminal pro-peptide (P1NP), a bone formation marker, decreased within 3 days of fasting. Whereas dual-energy x-ray absorptiometry measures of bone mineral density were unchanged after 10 days of fasting, high-resolution peripheral quantitative CT demonstrated remodeling of bone microarchitecture. Pathway analysis of longitudinal metabolomics data identified one-carbon metabolism as fasting dependent. In cultured osteoblasts, we tested the functional significance of one-carbon metabolites modulated by fasting, finding that methionine - which surged after 3 days of fasting - affected markers of osteoblast cell state in a concentration-dependent manner, in some instances exhibiting a U-shaped response with both low and high concentrations driving putative antibone responses. Administration of methionine to mice for 5 days recapitulated some fasting effects on bone, including a reduction in serum P1NP. In conclusion, a 10-day fast in humans led to remodeling of bone microarchitecture, potentially mediated by a surge in circulating methionine. These data support an emerging model that points to a window of optimal methionine exposure for bone health.

Transcriptional synergy in human aortic endothelial cells is vulnerable to combination p300/CBP and BET bromodomain inhibition.

Combinatorial signaling by proinflammatory cytokines synergizes to exacerbate toxicity to cells and tissue injury during acute infections. To explore synergism at the gene-regulatory level, we investigated the dynamics of transcription and chromatin signaling in response to dual cytokines by integrating nascent RNA imaging mass spectrometry, RNA sequencing, amplification-independent mRNA quantification, assay for transposase-accessible chromatin using sequencing (ATAC-seq), and transcription factor profiling. Costimulation with interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) synergistically induced a small subset of genes, including the chemokines CXCL9, -10, and -11. Gene induction coincided with increased chromatin accessibility at non-coding regions enriched for p65 and STAT1 binding sites. To discover coactivator dependencies, we conducted a targeted chemogenomic screen of transcriptional inhibitors followed by modeling of inhibitor dose-response curves. These results identified high efficacy of either p300/CREB-binding protein (CBP) or bromodomain and extra-terminal (BET) bromodomain inhibitors to disrupt induction of synergy genes. Combination p300/CBP and BET bromodomain inhibition at half-maximal inhibitory concentrations (subIC50) synergistically abrogated IFNγ/TNFα-induced chemokine gene and protein levels.

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 TGFß-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.

Evidence for ~12-h ultradian gene programs in humans.

Mice and many marine organisms exhibit ~12-h ultradian rhythms, however, direct evidence of ~12-h ultradian rhythms in humans is lacking. Here, we performed prospective, temporal transcriptome profiling of peripheral white blood cells from three healthy humans. All three participants independently exhibited robust ~12-h transcriptional rhythms in molecular programs involved in RNA and protein metabolism, with strong homology to circatidal gene programs previously identified in Cnidarian marine species.

Elimination of 15N-thymidine after oral administration in human infants.

BACKGROUND: We have developed a new clinical research approach for the quantification of cellular proliferation in human infants to address unanswered questions about tissue renewal and regeneration. The approach consists of oral 15N-thymidine administration to label cells in S-phase, followed by Multi-isotope Imaging Mass Spectrometry for detection of the incorporated label in cell nuclei. To establish the approach, we performed an observational study to examine uptake and elimination of 15N-thymidine. We compared at-home label administration with in-hospital administration in infants with tetralogy of Fallot, a form of congenital heart disease, and infants with heart failure. METHODS: We examined urine samples from 18 infants who received 15N-thymidine (50 mg/kg body weight) by mouth for five consecutive days. We used Isotope Ratio Mass Spectrometry to determine enrichment of 15N relative to 14N (%) in urine. RESULTS/FINDINGS: 15N-thymidine dose administration produced periodic rises of 15N enrichment in urine. Infants with tetralogy of Fallot had a 3.2-fold increase and infants with heart failure had a 4.3-fold increase in mean peak 15N enrichment over baseline. The mean 15N enrichment was not statistically different between the two patient populations (p = 0.103). The time to peak 15N enrichment in tetralogy of Fallot infants was 6.3 ± 1 hr and in infants with heart failure 7.5 ± 2 hr (mean ± SEM). The duration of significant 15N enrichment after a dose was 18.5 ± 1.7 hr in tetralogy of Fallot and in heart failure 18.2 ± 1.8 hr (mean ± SEM). The time to peak enrichment and duration of enrichment were also not statistically different (p = 0.617 and p = 0.887). CONCLUSIONS: The presented results support two conclusions of significance for future applications: (1) Demonstration that 15N-thymidine label administration at home is equivalent to in-hospital administration. (2) Two different types of heart disease show no differences in 15N-thymidine absorption and elimination. This enables the comparative analysis of cellular proliferation between different types of heart disease.

Dietary intake and glutamine-serine metabolism control pathologic vascular stiffness.

Perivascular collagen deposition by activated fibroblasts promotes vascular stiffening and drives cardiovascular diseases such as pulmonary hypertension (PH). Whether and how vascular fibroblasts rewire their metabolism to sustain collagen biosynthesis remains unknown. Here, we found that inflammation, hypoxia, and mechanical stress converge on activating the transcriptional coactivators YAP and TAZ (WWTR1) in pulmonary arterial adventitial fibroblasts (PAAFs). Consequently, YAP and TAZ drive glutamine and serine catabolism to sustain proline and glycine anabolism and promote collagen biosynthesis. Pharmacologic or dietary intervention on proline and glycine anabolic demand decreases vascular stiffening and improves cardiovascular function in PH rodent models. By identifying the limiting metabolic pathways for vascular collagen biosynthesis, our findings provide guidance for incorporating metabolic and dietary interventions for treating cardiopulmonary vascular disease.