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Research Project: The Microbiome as a Mediator of Host-Genome-Determined Lactation Outcomes and the Liver-Gut Axis in Lactation

Location: Children's Nutrition Research Center

2023 Annual Report

Objective 1: Select inbred mouse strains with phenotypic extremes in milk production will be used to: a) identify genomic variants along with intestinal and mammary-expressed genes that differentiate low and high milk production, and b) determine the extent to which genome-driven differences in milk production and mammary gene expression are directly mediated through host-dependent differences in the intestinal and/or mammary tissue microbiome. Subobjective 1A: Sequence the genomes of additional unsequenced strains from our original milk yield cohort and then use this completed lactation phenome genotype data to identify strain-specific private alleles and predict the functional consequences of these variants on genes with the potential to regulate traits defined in the lactation phenome dataset. Subobjective 1B: Combine the lactation phenome dataset with the expanded common variant data from sub objective 1A to conduct an enhanced joint-GWAS of SNP, INDEL, and SV, and to subsequently predict the functional consequences of the newly identified variants to lactation. Subobjective 1C: Using a complete 3x3 diallele cross of QSi3, QSi5, and PL/J determine the contribution of strain-dosage, heterosis, parent-of-origin, and epistasis to milk production and composition, and mammary gland development during early lactation, and identify mammary epithelial cell and intestinal eGenes on the basis of allelic imbalance. Subobjective 1D: Integrate the set of eGenes discovered in 1C with the set of private and common variants discovered 1A and1B and employ network modeling to predict and test those variant-eGene pairs that are most likely to cause the variation in the lactation phenome traits. Subobjective 1E: Analyze the fecal microbiota along with prolactin and oxytocin in samples obtained from the diallel conducted under sub-objective 1C to determine the contribution of strain-dosage, heterosis, parent-of-origin, and epistasis to the diversity and richness of the intestinal microbiota, to the abundance of specific taxa, and to neuroendocrine function in mouse strains with a genetic propensity for high or low milk yield. Objective 2: Determine the short and long-term impact of lactation on the maternal hepatic metabolome composition and hepatic signaling pathways in mice. Objective 3: Determine the impact of maternal nuclear receptor signaling on the maternal hepatic metabolome and pup viability.

Genetic background is known to influence variation in milk production however environmental factors also play a role. Advances in high-throughput DNA sequencing technologies have revolutionized the way in which the microbial world is viewed and has led to the concept that the microbiome is a major regulator of normal development and health. The microbiome is regulated by diet, but is also under the control of the host genome. In this regard, the full number of host genetic variants associated with lactation-related traits remains to be determined. Differences in milk production are driven by changes in gene expression within organs important to milk synthesis. Additionally, the intestinal microbiome is controlled by the host genome, but can directly influence gene expression within the host. We aim to understand how variations in the maternal genome interact with the microbiome to determine lactation success. Whole genome sequence data from select mouse strains will be used to identify genetic variants that are unique to high or low milk production. These newly identified variants will be functionally linked to milk production and composition, and to lactation-induced intestinal and mammary gene expression through a specific RNA Sequencing test known as allelic imbalance. Strain- and allele-dependent differences in fecal ribosomal 16s sequencing reads will associate the variants with the intestinal microbiome. Lastly, maternal microbiome seeding through neonatal cross-fostering will establish the ability of the intestinal microbiome to over-ride the effects of genetic background lactation-dependent gene expression and milk production. Additionally, although rates of breastfeeding (BF) have increased, there is much variability in BF initiation and duration rates. Lactation insufficiency, inability to produce enough breast milk to support offspring development, is estimated to be between 40-60%. The underlying mechanisms of lactation insufficiency are not well understood and require more study. The liver and small intestine undergo metabolic changes that support the production of mature milk in the mammary gland in lactating rodents, including significant increases in hepatic and intestinal bile acids. In lactating animal models, key enzymes involved in cholesterol and lipid homeostasis are altered during lactation. Bile acids promote the solubilization of cholesterol and lipid soluble nutrients, which enhance milk lipid nutrient composition. These genes are regulated by a group of transcription factors called nuclear receptors- the metabolic nuclear receptors farnesoid x receptor (FXR) and peroxisome proliferator activated receptor alpha (PPARalpha). These nuclear receptors and their target genes represent novel targets for study to address our central hypothesis that manipulation of hepatic and intestinal nuclear receptors alters lipid composition in breast milk. The overall goal of this project is to determine the role of FXR and PPARalpha in the metabolic adaptations of the maternal liver-gut axis.

Progress Report
In Objective 1, we used select inbred mouse strains with extremes in milk production to: a) identify genomic variants along with intestinal and mammary-expressed genes that differentiate low and high milk production, and b) determine the extent to which genome-driven differences in milk production and mammary gene expression are directly mediated through differences in the micro-organisms that live in intestinal and/or mammary tissue. Last year, we finished identifying DNA sequence variants in the mouse genome responsible for changes in milk fatty acid composition. There were 39,474 unique DNA sequence variants that were distributed among 164 sites across the genome. Of these variants, 25,941 were found within or near genes. There was a total of 5,041 genes found within regions of the genome containing one or more fatty acids associated sequence variant. Of these there were 606 genes identified of which 578 encoded for proteins, 12 were gene models with no known function, and 9 were non-coding RNA genes. By filtering these genes against a public dataset on lactation-dependent gene expression we narrowed our list of candidate genes to 404. With this list of lactation-dependent, mammary expressed genes, we ran gene ontology and pathway analysis through an on-line web tool known as "InnateDB". Through this work we identified 16 pathways that were over-represented. These overrepresented pathways were surprisingly not linked directly to lipid metabolism but were involved with the regulation of the immune response. A number of the genes in these pathways were involved in the body's ability to distinguish itself from non-self. Although pathways linked to fat metabolism were not statistically significant in over-representation from our gene list above, there were fat metabolism genes present. Among the list of mammary expressed genes there were 21 genes that are known to be important for fat metabolism. Of these 13 were enzymes, 7 were regulators of gene transcription, and 2 were hormone signaling molecules. Of these fat metabolism genes, we are now focusing our attention on 3. The gene "Olah" encodes an enzyme that is important for lactating mammary cells to make the large quantities of fatty acid molecules that make up milk fat. The gene "Mecr" encodes for an enzyme that sets the ability of the mammary cell mitochondria to make fat. This mitochondrial fat is important for determining how much energy the mammary cell can use to make milk. Lastly, the gene "Ppargc1a" encodes for a regulator of gene transcription that is in the cell nucleus and is also important to regulating the ability of mammary cells to use energy. Among our mammary expressed milk fatty acid associated genes there was also a set of 13 genes that are important for membrane transport of small molecules and 2 of these, "Slc25a48" and "Slc43a3", are important for the transport of fatty acids in the mammary cell. A second area for work conducted last year involved the completion of an animal experiment that measured milk production in the offspring of genetic crosses between three different families of mice that are divergent in milk production. We collected milk, mammary, intestine, and fecal samples from these mice. We used specialized software to partition variation in milk yield to maternal genetics versus other sources. We also isolated mammary RNA from these animals and did RNA sequencing but found that the ability to map strain-specific mammary-express genes did not produce reliable results. Because of the limited success with our diallel RNA sequencing we diverted our efforts to isolating mammary RNA from all 31 of the families in our mouse lactation study. Total mammary RNA was prepared from 155 lactating females with a sample size of 5 females for each of the families. We then used the standard mouse genome reference to map and count the number of lactation-dependent mammary-expressed mRNA transcripts that were present in the samples. This effort identified 55,297 transcripts in total and 13,684 expressed at a high enough level in the lactating mammary gland to make their measurement reliable. We are now comparing their expression among the different mouse families and will further use these results to locate regions in the mouse genome referred to as expression quantitative trait loci. We also know from this work that several of the fatty acid genes described above are differentially expressed between different mouse families. This further supports the idea that the genes Olah, Mecr, and Ppargc1a will play an important role in regulating milk fat synthesis and possibly other aspects of milk production. Lactation is a physiological state that exerts profound increases in energy demand and metabolic adaptation to meet nutritional requirements for the mother and offspring. Although there is a positive association of breastfeeding (BF) with wellness and health for both mother and offspring, there is very limited data regarding the maternal metabolic adaptations during lactation. Essential metabolic processes in the liver are regulated by the expression of genes controlled by a group of transcription factors known as nuclear receptors, in particular farnesoid x receptor (FXR) and peroxisome proliferating activated receptor alpha (PPARa). Our Objectives 2 and 3 focus on changes in metabolic gene expression of the liver and intestine in mice as described below, because these organ systems are essential to metabolic regulation. In regards to Objective 2, following establishment of the wild-type (control) group of mice, we completed initial tissue collection to test changes in the signaling pathways from lactating dams day 14 post-partum (the phase of lactation with maximal milk production) and whether changes in mice that had pups removed by day 1 post-partum (non-lactating mice 14 days post-partum (NL-PP14)) had similar metabolic gene signatures as lactating dams 14 days post-partum (L-PP14). Metabolic gene expression in NL-PP14 and L-PP14 were compared with the virgin controls. Lipogenic gene expression was increased in the both NL-PP14 and L-14 livers, suggesting that elevated lipogenic gene expression observed in lactating mice (L-PP14) is independent of the major lactogenic hormone prolactin. Triglyceride and very-low-density lipoprotein (VLDL) concentrations were lower in the L-PP14 mice (NL-PP14 was not measured), and future studies will determine lipid and fat composition in both serum and liver of virgin controls, NL-PP14, and L-PP14 mice. We also found increased expression of key genes required for the homeostasis of bile acid metabolism in the livers and intestine of both NL-PP14 and L-PP14 mice, as well as increased bile acids in the livers of L-PP14 mice. Increases in bile acids would support absorption of lipid-soluble nutrients. Surprisingly, hepatic genes involved in glucose homeostasis were increased in the NL-PP14 but not L-PP14 relative with virgin mice; however, we found decreased levels of glucose and other glycolytic intermediates in the livers of L-PP14 relative with virgin mice. We are continuing these studies in mice at postpartum day 2 (early lactation) and post-weaning and are collecting tissues from our additional mouse strains: metabolic nuclear receptor knockouts (FXR) and mice genetically modified to develop extreme liver accumulation of copper (Atp7b knockout).

1. Maternal metabolic adaptation during lactation. During pregnancy, the energy needs of the mother are maintained despite an increased need to support the growing fetus. Yet after birth, a dramatic shift to a negative energy balance (energy expenditure exceeds energy intake) occurs during the lactation period. A limited body of knowledge exists that addresses how breastfeeding metabolism changes during lactation. Researchers at the Children’s Nutrition Research Center in Houston, Texas, studied key metabolic regulators in the liver and intestine of lactating and non-lactating mice. We found several regulatory factors in lipid metabolism were increased during lactation, indicating metabolic adaptations occur and potential long-term metabolic consequences from lactation were also identified. This lactation study provides valuable insights in the body’s adaptation to changes in metabolic needs, sustaining healthy nutrition for the mother and offspring, and the potential long-term metabolic benefits for the mother.