Early life exposure to vitamin D deficiency impairs molecular mechanisms that regulate liver cholesterol biosynthesis, energy metabolism, inflammation, and detoxification.

Introduction: Emerging data suggests liver disease may be initiated during development when there is high genome plasticity and the molecular pathways supporting liver function are being developed. Methods: Here, we leveraged our Collaborative Cross mouse model of developmental vitamin D deficiency (DVD) to investigate the role of DVD in dysregulating the molecular mechanisms underlying liver disease. We defined the effects on the adult liver transcriptome and metabolome and examined the role of epigenetic dysregulation. Given that the parental origin of the genome (POG) influences response to DVD, we used our established POG model [POG1-(CC011xCC001)F1 and POG2-(CC001xCC011)F1] to identify interindividual differences. Results: We found that DVD altered the adult liver transcriptome, primarily downregulating genes controlling liver development, response to injury/infection (detoxification & inflammation), cholesterol biosynthesis, and energy production. In concordance with these transcriptional changes, we found that DVD decreased liver cell membrane-associated lipids (including cholesterol) and pentose phosphate pathway metabolites. Each POG also exhibited distinct responses. POG1 exhibited almost 2X more differentially expressed genes (DEGs) with effects indicative of increased energy utilization. This included upregulation of lipid and amino acid metabolism genes and increased intermediate lipid and amino acid metabolites, increased energy cofactors, and decreased energy substrates. POG2 exhibited broader downregulation of cholesterol biosynthesis genes with a metabolomics profile indicative of decreased energy utilization. Although DVD primarily caused loss of liver DNA methylation for both POGs, only one epimutation was shared, and POG2 had 6.5X more differentially methylated genes. Differential methylation was detected at DEGs regulating developmental processes such as amino acid transport (POG1) and cell growth & differentiation (e.g., Wnt & cadherin signaling, POG2). Conclusions: These findings implicate a novel role for maternal vitamin D in programming essential offspring liver functions that are dysregulated in liver disease. Importantly, impairment of these processes was not rescued by vitamin D treatment at weaning, suggesting these effects require preventative measures. Substantial differences in POG response to DVD demonstrate that the parental genomic context of exposure determines offspring susceptibility.

Timing of standard chow exposure determines the variability of mouse phenotypic outcomes and gut microbiota profile.

Standard chow diet contributes to reproducibility in animal model experiments since chows differ in nutrient composition, which can independently influence phenotypes. However, there is little evidence of the role of timing in the extent of variability caused by chow exposure. Here, we measured the impact of diet (5V5M, 5V0G, 2920X, and 5058) and timing of exposure (adult exposure (AE), lifetime exposure (LE), and developmental exposure (DE)) on growth & development, metabolic health indicators, and gut bacterial microbiota profiles across genetically identical C57BL6/J mice. Diet drove differences in macro- and micronutrient intake for all exposure models. AE had no effect on measured outcomes. However, LE mice exhibited significant sex-dependent diet effects on growth, body weight, and body composition. LE effects were mostly absent in the DE model, where mice were exposed to chow differences from conception to weaning. Both AE and LE models exhibited similar diet-driven beta diversity profiles for the gut bacterial microbiota, with 5058 diet driving the most distinct profile. Diet-induced beta diversity profiles were sex-dependent for LE mice. Compared to AE, LE drove 9X more diet-driven differentially abundant genera, majority of which were the result of inverse effects of 2920X and 5058. Our findings demonstrate that lifetime exposure to different chow diets has the greatest impact on reproducibility of experimental measures that are common components of preclinical mouse model studies. Importantly, weaning DE mice onto a uniform diet is likely an effective way to reduce unwanted phenotypic variability among experimental models.

Highlights from the 24th workshop on vitamin D in Austin, September 2022.

The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.

Confirmation of high-throughput screening data and novel mechanistic insights into FXR-xenobiotic interactions by orthogonal assays.

Toxicology in the 21st Century (Tox21) is a federal collaboration employing a high-throughput robotic screening system to test 10,000 environmental chemicals. One of the primary goals of the program is prioritizing toxicity evaluations through in vitro high-throughput screening (HTS) assays for large numbers of chemicals already in commercial use for which little or no toxicity data is available. Within the Tox21 screening program, disruption in nuclear receptor (NR) signaling represents a particular area of interest. Given the role of NR's in modulating a wide range of biological processes, alterations of their activity can have profound biological impacts. Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that has demonstrated importance in bile acid homeostasis, glucose metabolism, lipid homeostasis and hepatic regeneration. In this study, we re-evaluated 24 FXR agonists and antagonists identified through Tox21 using select orthogonal assays. In transient transactivation assays, 7/8 putative agonists and 4/4 putative inactive compounds were confirmed. Likewise, we confirmed 9/12 antagonists tested. Using a mammalian two hybrid approach we demonstrate that both FXR agonists and antagonists facilitate FXRα-coregulator interactions suggesting that differential coregulator recruitment may mediate activation/repression of FXRα mediated transcription. Additionally, we tested the ability of select FXR agonists and antagonists to facilitate hepatic transcription of FXR gene targets Shp and Bsep in a teleost (Medaka) model. Through application of in vitro cell-based assays, in silico modeling and in vivo gene expressions, we demonstrated the molecular complexity of FXR:ligand interactions and confirmed the ability of diverse ligands to modulate FXRα, facilitate differential coregulator recruitment and activate/repress receptor-mediated transcription. Overall, we suggest a multiplicative approach to assessment of nuclear receptor function may facilitate a greater understanding of the biological and mechanistic complexities of nuclear receptor activities and further our ability to interpret broad HTS outcomes.

Correction: Kassotis et al. Nonylphenol Polyethoxylates Enhance Adipose Deposition in Developmentally Exposed Zebrafish. Toxics 2022, 10, 99.

In the original publication [...].

Nonylphenol Polyethoxylates Enhance Adipose Deposition in Developmentally Exposed Zebrafish.

Alkylphenol polyethoxylates (APEOs), such as nonylphenol ethoxylates (NPEOs), are high-production-volume surfactants used in laundry detergents, hard-surface cleaners, pesticide formulations, textile production, oils, paints, and other products. NPEOs comprise -80% of the total production of APEOs and are widely reported across diverse environmental matrices. Despite a growing push for replacement products, APEOs continue to be released into the environment through wastewater at significant levels. Research into related nonionic surfactants from varying sources has reported metabolic health impacts, and we have previously demonstrated that diverse APEOs and alcohol polyethoxylates promote adipogenesis in the murine 3T3-L1 pre-adipocyte model. These effects appeared to be independent of the base alkylphenol and related to the ethoxylate chain length, though limited research has evaluated NPEO exposures in animal models. The goals of this study were to assess the potential of NPEOs to promote adiposity (Nile red fluorescence quantification) and alter growth and/or development (toxicity, length, weight, and energy expenditure) of developmentally exposed zebrafish (Danio rerio). We also sought to expand our understanding of the ability to promote adiposity through evaluation in human mesenchymal stem cells. Herein, we demonstrated consistent adipogenic effects in two separate human bone-marrow-derived mesenchymal stem cell models, and that nonylphenol and its ethoxylates promoted weight gain and increased adipose deposition in developmentally exposed zebrafish. Notably, across both cell and zebrafish models we report increasing adipogenic/obesogenic activity with increasing ethoxylate chain lengths up to maximums around NPEO-6 and then decreasing activity with the longest ethoxylate chain lengths. This research suggests metabolic health concerns for these common obesogens, suggesting further need to assess molecular mechanisms and better characterize environmental concentrations for human health risk assessments.

Vitamin D deficiency promotes accumulation of bioactive lipids and increased endocannabinoid tone in zebrafish.

Vitamin D is well known for its traditional role in bone mineral homeostasis; however, recent evidence suggests that vitamin D also plays a significant role in metabolic control. This study served to investigate putative linkages between vitamin D deficiency (VDD) and metabolic disruption of bioactive lipids by MS imaging. Our approach employed infrared-matrix-assisted laser desorption electrospray ionization MS imaging for lipid metabolite profiling in 6-month-old zebrafish fed either a VDD or a vitamin D-sufficient (VDS) diet. Using a lipidomics pipeline, we found that VDD zebrafish had a greater abundance of bioactive lipids (N-acyls, endocannabinoids [ECs], diacylglycerols/triacylglycerols, bile acids/bile alcohols, and vitamin D derivatives) suggestive of increased EC tone compared with VDS zebrafish. Tandem MS was performed on several differentially expressed metabolites with sufficient ion abundances to aid in structural elucidation and provide additional support for MS annotations. To confirm activation of the EC pathways, we subsequently examined expression of genes involved in EC biosynthesis, metabolism, and receptor signaling in adipose tissue and liver from VDD and VDS zebrafish. Gene expression changes were congruent with increased EC tone, with VDD zebrafish demonstrating increased synthesis and metabolism of anandamide compared with VDS zebrafish. Taken together, our data suggest that VDD may promote accumulation of bioactive lipids and increased EC tone in zebrafish.

Vitamin D deficiency serves as a precursor to stunted growth and central adiposity in zebrafish.

Emerging evidence demonstrates the importance of sufficient vitamin D (1α, 25-dihydroxyvitamin D3) levels during early life stage development with deficiencies associated with long-term effects into adulthood. While vitamin D has traditionally been associated with mineral ion homeostasis, accumulating evidence suggests non-calcemic roles for vitamin D including metabolic homeostasis. In this study, we examined the hypothesis that vitamin D deficiency (VDD) during early life stage development precedes metabolic disruption. Three dietary cohorts of zebrafish were placed on engineered diets including a standard laboratory control diet, a vitamin D null diet, and a vitamin D enriched diet. Zebrafish grown on a vitamin D null diet between 2-12 months post fertilization (mpf) exhibited diminished somatic growth and enhanced central adiposity associated with accumulation and enlargement of visceral and subcutaneous adipose depots indicative of both adipocyte hypertrophy and hyperplasia. VDD zebrafish exhibited elevated hepatic triglycerides, attenuated plasma free fatty acids and attenuated lipoprotein lipase activity consistent with hallmarks of dyslipidemia. VDD induced dysregulation of gene networks associated with growth hormone and insulin signaling, including induction of suppressor of cytokine signaling. These findings indicate that early developmental VDD impacts metabolic health by disrupting the balance between somatic growth and adipose accumulation.

Methods for Cryosectioning and Mass Spectrometry Imaging of Whole-Body Zebrafish.

The zebrafish (Danio rerio) is an ideal model for whole animal studies of lipid metabolism and lipid-related disease. In this work, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) was applied for direct visualization of lipid and metabolite distributions across various organs in whole-body zebrafish tissue sections. Detailed methods for overcoming the challenges of cryosectioning adult male zebrafish for MSI and complementary histological imaging are described. Representative two-dimensional ion maps demonstrated organ specific localization of lipid analytes allowing for visualization of areas of interest including the brain, liver, intestines, and skeletal muscle. A high resolving power mass spectrometer was utilized for accurate mass measurements, which permitted the use of open-source, web-based tools for MS1 annotations including METASPACE and METLIN. Whole-body MSI with IR-MALDESI allowed for broad lipid coverage with high spatial resolution, illustrating the potential of this technique for studying lipid-related diseases using zebrafish as a model organism.