Perinatal Caprine Milk Oligosaccharides Consumption: Altering the Maternal and Offspring Liver Gene Expression

Abstract

Consumption of Caprine Milk Oligosaccharides (CMO) by dams during gestation and lactation, compared to a control diet or a diet supplemented with Galacto-oligosaccharides (GOS), was associated with decreased maternal liver weight, increased offspring body weight and length at weaning, and increased offspring visceral fat and serum leptin concentration 30 days after weaning. These changes suggest that dietary CMO alters lipid metabolism, both in dams and offspring. We hypothesized that perinatal CMO intake affected expression of maternal genes in the liver involved in energy metabolism, and programmed pups’ metabolic function leading to increased post-weaning visceral adiposity. To characterise the effects of perinatal consumption of CMOs on maternal and offspring liver gene expression, C57BL/6 mice were fed either a control, CMO, or GOS diet from mating to weaning. From weaning, half of the pups from each maternal group were fed the control diet for 30 days. Microarray analysis was conducted on liver samples from dams and offspring. Differences in the expression of genes involved in lipid metabolism were observed in dams and changes in expression of hepatic genes involved in energy balance and steroid metabolism were observed in pups at weaning. Increased visceral fat was observed in pups 30 days. Perinatal consumption of CMO diet affected infant lipid metabolism, which may be related to altered expression of genes in the liver involved in energy balance and lipid metabolism in dams.

Keywords

Caprine milk oligosaccharides; Perinatal; Liver; Microarray; Lipid metabolism; Visceral fat

Introduction

Caprine Milk Oligosaccharides (CMO) were shown to support the growth of gut beneficial bacteria in vitro studies [1,2] revealing CMO had potentially had prebiotic effects. Using a mouse model, the consumption of CMO by dams during gestation and lactation, compared to the consumption of a control diet or diet supplemented with Galacto-Oligosaccharides (GOS), was associated with changes in the maternal colon microbiota, and in milk composition, which affected pup development [3]. The specific findings related to CMO consumption by dams included (i) increased milk protein concentration; (ii) decreased liver weight in dams; (iii) increased colon length in dams and pups at weaning; (iv) increased body weight and length and proportions of Bifidobacterium in the colon of pups at weaning; and iv) increased body visceral fat and serum leptin concentration in pups 30 days after weaning. These findings highlight the potential for indirect effects of food components in the maternal diet on offspring development, Gastrointestinal Tract (GIT) composition, and metabolism.

Changes in colon microbiota and increased body fat and serum leptin concentration observed in dams and pups after maternal consumption of CMO may indicate that CMO altered lipid metabolism, both in dams and offspring. Although no difference was found in dams’ body weight or visceral fat, a decrease in liver weight may indicate decreased fat accumulation in this organ [4] and/or altered lipid metabolism [5]. Altered lipid metabolism could also be observed in pups 30 days after weaning, which had an increase in visceral body fat and leptin serum concentration compared to pups from dams fed GOS or a control diet [3]. Here we describe liver gene expression in the maternal and pup’s samples from the same study.

Materials and Methods

Animals and study design

As previously described [3], animal experimentation was approved by AgResearch Grasslands Animal Ethics Committee (Application No. AE12579), in accordance with the New Zealand Animal Welfare Act 1999, New Zealand. In short, sixty-three C57BL/6 mice (42 female and 21 male) were obtained from the AgResearch Ruakura Small Animal Facility, Hamilton, New Zealand. Nine-week-old mice were randomly assigned to groups of two females and one male and fed one of the following diets; AIN-76A (control diet), AIN-76A supplemented with 1% GOS (GOS diet), or AIN-76A supplemented with Caprine Milk Oligosaccharide Fraction (CMOF) containing 1% caprine milk oligosaccharides (CMO diet). Diets were formulated by Research Diets, Inc. (NJ, USA). The CMO diet also contained 0.2% of GOS and other sugars as a result of the method used to obtain the CMOF (Table 1). To ruled out the effect of GOS on maternal and offspring health outcomes 1% GOS was supplemented to the control diet. CMOF was obtained by a previously described method [6].

Table 1. Composition of diets and caprine milk oligosaccharide enriched fraction.

After delivery, pups remained with their dams up to weaning (21 days) where dams and half of the pups were euthanized by CO2 and cervical dislocation and sampled. To determine the longer-term effects of maternal diet, the remaining pups were fed the control diet for 30 days, then euthanized. The left lobe of liver was collected from each animal and kept at -80ºC until analysis. Complete experimental methods and results were previously described in Thum, et al (2016) [3].

Liver RNA extraction

Gene expression profiles in the liver samples (n=4 animals per treatment per age group) was measured using Agilent Mouse Gene Expression 8 × 60K microarrays (catalog number G4852A; Agilent Technologies, California, USA), with each array consisting of 60,000 sequence probes, which map to 39,430 unique Entrez Gene identifiers, and 32974 unique gene symbols [7].

RNA and protein were extracted from liver tissue using the Qiagen AllPrep DNA/RNA/Protein Mini Kit according to the manufacturer’s instructions (Qiagen Inc., California, USA). The RNA integrity was assessed using a RNA6000 Nano Labchip kit with an Agilent 2100 Bioanalyzer (Agilent Technologies), and only RNA samples with an RNA Integrity Number (RIN) greater than 6 used for subsequent analysis.

Extracted sample RNA and reference RNA were transcribed into cRNA and simultaneously labelled with cyanine dyes Cy3 or Cy5 according to the two-colour Quick Amp Labelling kit (Agilent Technologies). RNA that met quality criteria was transcribed into complementary RNA (cRNA) using T7 RNA polymerase and simultaneously amplified and labelled with the green fluorescent dye Cy3 or red fluorescent dye Cy5, in a dye-swap design, to avoid dye bias effect. The reference RNA used was extracted from intestine, kidney, liver and foetus of Swiss mice. The quality of the amplification and incorporation of the dye were assessed using a NanoDrop ND-1000 spectrophotometer, and only samples with a cRNA yield greater than 825 ng and a specific activity greater than 8 pmol Cy3 or Cy5 per μg cRNA were used. RNA Spike-in kits were included as instructed by the manufacturer (Agilent Technologies, California, USA).

Samples were hybridised onto Agilent Mouse Gene Expression 8 × 60K microarrays using four biological replicates per treatment/age, with 1 hybridisation for each biological replicate. Gene expression hybridisation kits were used as instructed by manufacturer (Agilent Technologies).

Microarray data analysis

Microarray data were processed using the Bioconductor suite of packages in R [8]. Differentially expressed genes were determined using an empirical Bayes modified t-statistic with the limma package [9]. Genes with a>1.5-fold-change (FC) and a Benjamini and Hochberg false-discovery rate adjusted P-value (q)<0.05 were considered to be differentially expressed. Gene Ontology functions of the differentially expressed mapped genes were identified using the GeneMANIA [10] plugin for Cytoscape [11] using default settings. Additionally, Gene Set Enrichment Analysis (GSEA) was performed using the limma package in R on pathways listed in the Reactome curated database [12] (http://www.reactome.org/), with P<0.05 considered significant. It is important to note that, although validation of microarray data by qPCR is widely thought to be required, the reliability of using one technology to confirm the results of the other presents its own limitations. Nevertheless, it has been repeatedly demonstrated that the two methods yield comparable results, making repeated validation unnecessary. Furthermore, qPCR cannot be used to assess or confirm results from methods such as GSEA, which examine pathway-wide transcriptional changes.

Results

Dams fed the CMO, or GOS diet had 119 and 133 differentially expressed unique genes in the liver, respectively, compared to dams fed the control diet (Figure 1). The effects of maternal diet were also observed in the pups liver gene transcription where pups at weaning from dams fed CMO and GOS had 109 and 85 genes differently expressed, respectively. The longer-term effects of maternal diets were less prominent in pups; after they consumed the control diet for 30 days, only 7 and 2 liver genes were differently expressed in pups from dams fed the CMO and GOS diet, respectively (Figure 1).

Figure 1: Number of genes with a significant change in expression by each treatment/age (n=4 per treatment per age group). Note: Decreased; Increased.

Differentially expressed genes in dams fed the CMO diet compared to dams fed the control diet (Table 2, Supplementary material Figure 1) were primarily involved in the regulation of lipid metabolism or ubiquitination and SUMOylation (addition of a small ubiquitin-like modifier).

Table 2. Genes differently expressed in dams fed CMO compared to control diet.

At weaning, genes involved in the regulation of mitochondrial membrane permeability in apoptotic processes, cytochrome P450 activity, and steroid metabolism were differentially expressed in the liver of pups from dams fed CMO compared to pups from dams fed the control diet (Table 3, Supplementary material Figure 2). By thirty days after weaning only a small number of liver genes were differentially expressed in pups from dams fed CMO compared to pups from dams fed the control diet (Table 4, Supplementary material Figure 3). Analysis of the pups’ liver transcriptome by GSEA (Table 5) indicated that the expression of genes involved in the synthesis of epoxy (EET) and dihydroxyeicosatrienoic acids (DHET) was increased by 25% and genes involved in collagen metabolism such as assembly of collagen fibrils (11%), collagen degradation (6%), collagen biosynthesis (6%) and formation (7%), and genes involved in extracellular matrix proteoglycans (6%) were down-regulated, in pups 30 after weaning from dams fed the CMO diet compared to pups from dams fed the control diet (Table 5).

Table 3. Genes differently expressed in pups at weaning from dams fed CMO compared to control diet (n=4 per treatment per age group).

Table 4. Genes differentially expressed in pups 30 days after weaning from dams fed CMO compared to pups from dams fed the control diet (n=4 per treatment per age group).

Table 5. GSEA pathway analysis of genes significantly changed in the liver of pups 30 days after weaning from dams fed CMO diet (n=4 per treatment per age group).

Discussion

Effect of CMO diet on maternal liver gene expression

This study showed that perinatal CMO intake affected expression of genes in the maternal liver related to energy and lipid metabolism compared to the control diet; changes included down-regulatation of genes such as Lcn2 (lipocalin 2) and Prkag1 (protein kinase AMP-activated non-catalytic subunit gamma). Reduction of Lcn2 (7.1 fold) and Prkag1 (1.5 fold), a subunit of 5' AMP-activated protein kinase, has been linked to inhibition of hepatic fatty acid oxidation and promotion of cholesterol synthesis, lipogenesis, and triglyceride synthesis [13,14]. Concentrations of lipids and other metabolites, however, were not confirmed by metabolomic or histological analysis of the liver.

Genes involved in ubiquitination and SUMOylation were also among differentially expressed transcripts in the liver of dams fed CMO compared to the control diet (Table 2). Post-translational modification of target protein substrates by ubiquitin-like proteins (Ubls) and SUMO proteins regulates cellular signaling in numerous processes such as metabolism, transcription, translation, vesicle transport and apoptosis [15,16]. Diet effects on the liver ubiquitome and its role in the regulation of whole-body euglycemia and lipidemia have recently been reported in rats [17]. More specifically, it was observed that ubiquitination of proteins is a key regulatory mechanism controlling fatty acid metabolism which may mediate the pathogenesis of fatty acid-associated diseases. Variations observed in this study on liver ubiquitination genes and genes involved in lipid metabolism combined with reduced liver weight suggest that CMO may have modified liver lipid metabolism towards increased energy absorption and utilization.

Effects of perinatal CMO consumption on offspring liver gene expression

Pups from dams fed the CMO diet, for example, had increased expression of two genes involved in steroid metabolism at weaning. The expression of both Hsd3b5 (3 beta-hydroxysteroid dehydrogenase type 5) and Srd5a1 (3-oxo-5α-steroid 4-dehydrogenase 1) has been shown to negatively correlate with lipid accumulation in liver [18,19]. Although no changes in liver weight and other markers for liver lipid metabolism were found at weaning in pups from dams fed CMO, increased body growth (weight and length) may be correlated with changes in gene expression observed in the liver. Concentration of blood lipids, however, were not evaluated in this study.

Other genes up-regulated in weaned pups from CMO-fed dams included those related to cytochrome P450s (CYP450) function. The effects of diet on CYP450 have been established and reviewed [20]. An increase in protein-to-carbohydrate ratio in the diet, for example, was shown to increase products of steroid hormone metabolism contributing to the transcriptional regulation of drug-metabolizing genes P450s [21]. Indeed, weaned pups from dams fed the CMO diet had increased intake of protein-to-carbohydrate ratio, as maternal milk had higher concentrations of protein compared to control fed dams [3].

By thirty days after weaning, pups from dams fed CMO compared to control diet, had no indication of expected changes in liver functionality, previously linked with increased visceral fat weight and serum leptin concentration [22]. Analysis of the pups’ liver transcriptome by GSEA, however, showed that the expression of genes involved in the synthesis of epoxy (EET) and dihydroxyeicosatrienoic acids (DHET) and collagen metabolism were increased.

The EETs are signaling molecules formed within various types of cells by the metabolism of arachidonic acid by a specific subset of CYP450 enzymes. The EETs have been most studied in animal models where they show the ability to prevent arterial occlusive diseases such as heart attacks and brain strokes by their anti-hypertensive and anti-inflammatory effects on blood vessels [23]. In the liver, collagens and proteoglycans play an intrinsic role in liver function in health and disease [24]. The interaction of collagens and proteoglycans provide architectural elements for the liver with basement membrane or other duct architecture.

Traditionally, visceral obesity is strongly associated with metabolic diseases [25], however, as observed here, no expected changes on liver metabolism linked to the onset of metabolic diseases were observed in pups 30 days after weaning. This may be due to non-pathological residual effects of metabolic changes observed in early life or may be explained by mouse strain variations in response to diet-induced obesity [26].

Conclusion

Perinatal consumption of the CMO diet affected maternal and offspring liver gene expression, with effects most prominent in dams and pups at weaning compared to pups 30 days after weaning, which had consumed the control diet. Differences in expression of ubiquitination and lipid metabolism-related genes in the dams’ liver combined with previously reported reductions in liver weight and changes in colon microbiota of the dams suggest that CMO may have affected maternal lipid metabolism towards increased energy absorption and utilization. These effects may have been manifested in the offspring resulting in increased body growth (body length and weight) as well as increasing the expression of genes involved in energy balance and steroid metabolism. The previously reported increase in visceral fat mass, observed post-weaning, is unlikely to be explained by changes in pups liver gene expression (which were minimal), but instead could be a residual effect of metabolic changes observed in dams and pups at weaning trigged by maternal CMO intake. To deeper understanding of the effects of CMO on maternal and offspring energy metabolism, more studies are needed linking gene expression, metabolomic data and liver histology.

Declarations

Ethics approval

Animal experimentation was approved by AgResearch Grasslands Animal Ethics Committee (Application No. AE12579), in accordance with the New Zealand Animal Welfare Act 1999, New Zealand.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by the Riddet Institute, a New Zealand Centre of Research Excellence and the Strategic Science Investment Fund (SSIF) from New Zealand Ministry of Business, Innovation and Employment.

Authorship

C.T., N.R., and W.M. designed the study. C.T. supported by W.Y. performed the experiment, analyzed the data, and wrote the paper. All authors proof-read the paper.

Acknowledgements

Not applicable.

References

1. Thum C, et al. In Vitro fermentation of caprine milk oligosaccharides by bifidobacteria isolated from breast-fed infants. Gut Microbes. 2015;6:352-363.

   [Crossref][Google Scholar][Pubmed]

2. Oliveira DL, et al. In vitro evaluation of the fermentation properties and potential prebiotic activity of caprine cheese whey oligosaccharides in batch culture systems. Biofactors. 2012;6:440-449.

   [Crossref][Google Scholar][Pubmed]

3. Thum C, et al. Prenatal caprine milk oligosaccharide consumption affects the development of mice offspring. Mol Nutr Food Res. 2016;60:2076-2085.

   [Crossref][Google Scholar][Pubmed]

4. Parnell JA, et al. Effect of prebiotic fibre supplementation on hepatic gene expression and serum lipids: A dose-response study in JCR: LA-cp rats. Br J Nutr. 2010;103:1577-1584.

   [Crossref][Google Scholar][Pubmed]

5. Nguyen P, et al. Liver lipid metabolism. J Anim Physiol Anim Nutr. 2008;92:272-283.

   [Crossref][Google Scholar][Pubmed]

6. Thum C, et al. Composition and enrichment of caprine milk oligosaccharides from New Zealand saneen goat cheese whey. J Food Comp Anal. 2015;42:30-37.

   [Crossref][Google Scholar]

7. Wheeler DL, et al. Database resources of the national center for biotechnology information. Nucleic Acids Res. 2005;33:39-45.

   [Crossref][Google Scholar][Pubmed]

8. Team C. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. 2016.
9. Ritchie ME, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47.

   [Crossref][Google Scholar][Pubmed]

10. Montojo J, et al. GeneMANIA cytoscape plugin: Fast gene function predictions on the desktop. Bioinformatics. 2010;26:2927-2928.

    [Crossref][Google Scholar][Pubmed]

11. Shannon P, et al. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498-2504.

    [Crossref][Google Scholar][Pubmed]

12. Ligtenberg W. A set of annotation maps for reactome. Reactome.

    [Crossref][Google Scholar]

13. Winder WW, et al. AMP-activated protein kinase, a metabolic master switch: Possible roles in type 2 diabetes. Am J Physiol. 1999;277:E1-E10.

    [Crossref][Google Scholar][Pubmed]

14. Zhang Y, et al. Lipocalin 2 is an important regulator of lipid metabolism and energy utilization. Nutrition. 2011;25:105.

    [Crossref][Google Scholar]

15. Callis J. The ubiquitination machinery of the ubiquitin system. Arabidopsis Book. 2014;12:e0174.

    [Crossref][Google Scholar][Pubmed]

16. Li J, et al. Desumoylase SENP6 maintains osteochondroprogenitor homeostasis by suppressing the p53 pathway. Nat Commun. 2018;9:143.

    [Crossref][Google Scholar][Pubmed]

17. Nagarajan SR, et al. Insulin and diet-induced changes in the ubiquitin-modified proteome of rat liver. PloS one. 2017;12:e0174431.

    [Crossref][Google Scholar][Pubmed]

18. Guillen N, et al. Microarray analysis of hepatic gene expression identifies new genes involved in steatotic liver. Physiol Genom. 2009;37:187-198.

    [Crossref][Google Scholar][Pubmed]

19. Nasiri M, et al. 5 α -reductase type 2 regulates glucocorticoid action and metabolic phenotype in human hepatocytes. Endocrinology. 2015;156:2863-2871.

    [Crossref][Google Scholar][Pubmed]

20. Anderson KE, et al. Dietary regulation of cytochrome P450. Annu Rev Nutr. 1991;11:141-167.

    [Crossref][Google Scholar][Pubmed]

21. Cherala G, et al. Effect of perinatal low protein diets on the ontogeny of select hepatic cytochrome p450 enzymes and cytochrome p450 reductase in the rat. Drug Metab Dispos. 2007;35:1057-1063.

    [Crossref][Google Scholar][Pubmed]

22. Renaud HJ, et al. Effect of diet on expression of genes involved in lipid metabolism, oxidative stress, and inflammation in mouse liver-insights into mechanisms of hepatic steatosis. PloS one. 2014;9:e88584.

    [Crossref][Google Scholar][Pubmed]

23. Spector AA, et al. Epoxyeicosatrienoic acids (EETs): Metabolism and biochemical function. Prog Lipid Res. 2004;43:55-90.

    [Crossref][Google Scholar][Pubmed]

24. Baiocchini A, et al. Extracellular matrix molecular remodeling in human liver fibrosis evolution. PLoS One. 2016;11:e0151736.

    [Crossref][Google Scholar][Pubmed]

25. Shuster A, et al. The clinical importance of visceral adiposity: A critical review of methods for visceral adipose tissue analysis. Br J Radiol. 2012;85:1-10.

    [Crossref][Google Scholar][Pubmed]

26. Montgomery M, et al. Mouse strain-dependent variation in obesity and glucose homeostasis in response to high-fat feeding. Diabetologia. 2013;56:1129-1139.

    [Crossref][Google Scholar][Pubmed]