Inhibition of proteolysis in histiotrophic nutrition pathways alters DNA methylation and one-carbon metabolism in the organogenesis-stage rat conceptus

Abstract

Epigenetic modifications, including DNA methylation, contribute to the transcriptional regulation of developmental genes that control growth and differentiation during embryogenesis. The methyl donor, S-adenosylmethionine (SAM) is biosynthesized from methionine and ATP by methionine adenosyltransferase 2a (Mat2a) in the one-carbon (C1) metabolism pathway. SAM biosynthesis requires a steady supply of nutrients, vitamins, and cofactors obtained by the developing conceptus through histiotrophic nutrition pathways (HNPs). The visceral yolk sac (VYS) captures proteins and their substrate cargos by receptor-mediated endocytosis (RME) and degrades them using lysosomal proteases. We hypothesize that leupeptin, a protease inhibitor, reduces the availability of methionine and C1 substrates, restricting SAM biosynthesis, and altering patterns of DNA methylation. Rat conceptuses were exposed to 50 and 100 μM leupeptin in whole embryo culture (WEC) for periods of 26 h from gestational day (GD) 10 or 6 h on GD11. after 6 h on GD11, the 100 μM leupeptin treatment significantly decreased methionine in embryo (EMB) and VYS, reduced Mat2a protein levels, and inhibited Mat2a specific activity; all of which produced a significant 52% reduction of SAM in the VYS. The 50 and 100 μM leupeptin treatments significantly decreased global methylation levels by 6–9% in EMB and 11–15% in VYS following both 6 and 26 h exposure periods. This study demonstrates that HNP disruption alters C1 activity and significantly reduces global DNA methylation during organogenesis. Because epigenetic reprogramming is crucial for normal differentiation and growth, these findings suggest a possible mechanism through which nutrients and environmental factors may alter early developmental regulation.

Mono-2-ethylhexyl phthalate (MEHP) alters histiotrophic nutrition pathways and epigenetic processes in the developing conceptus

“…Decreases in HNP function following treatment with the protease inhibitor leupeptin result in C 1 deficiencies 6 h after initial exposure [16]. Here, however, C 1 metabolic components follow an increasing trend with increasing MEHP concentrations in culture after 24 h. These results were unexpected because HNP activity is reduced in a dose-dependent fashion after 3 h, and it was believed that methyl donors and C 1 components should decrease initially.…”

“…Quantification of C 1 components was performed as previously described in [16]. Liquid chromatography-tandem mass spectrometry was performed using a TSQ Quantum Ultra AM triple quadrupole mass spectrometer (Thermo Scientific, Rockford, IL) coupled to a Waters 2695 Separations Module (Waters, Milford, MA).…”

“…Concentrations of each analyte were calculated by ratio of the measured analyte to the known quantity of the isotopic standard in each sample. All data was normalized to the total protein concentration of the conceptus to account for the differences in size and morphology due to MEHP treatment [16, 30]. …”

“…LUMA was utilized as explained in [38, 39] and previously performed in [16]. This is an extension assay which relies on differential cleavage of the DNA by enzymes that are sensitive or insensitive to methylation.…”

“…Because these processes occur during the phase of HNP-supplied nutrition and provide the necessary methyl donors for epigenetic marks, it is possible that perturbation of HNPs may result in abnormal epigenetic programming. Previous work has demonstrated that inhibition of HNP lysosomal function with leupeptin resulted in decreased DNA methylation in the EMB and VYS during early organogenesis [16]. …”