Paternal B Vitamin Intake Is a Determinant of Growth, Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring

Abstract

Background: The importance of maternal nutrition to offspring health and risk of disease is well established. Emerging evidence suggests paternal diet may affect offspring health as well.

Objective: In the current study we sought to determine whether modulating pre-conception paternal B vitamin intake alters intestinal tumor formation in offspring. Additionally, we sought to identify potential mechanisms for the observed weight differential among offspring by profiling hepatic gene expression and lipid content.

Methods: Male Apc1638N mice (prone to intestinal tumor formation) were fed diets containing replete (control, CTRL), mildly deficient (DEF), or supplemental (SUPP) quantities of vitamins B2, B6, B12, and folate for 8 weeks before mating with control-fed wild type females. Wild type offspring were euthanized at weaning and hepatic gene expression profiled. Apc1638N offspring were fed a replete diet and euthanized at 28 weeks of age to assess tumor burden.

Results: No differences in intestinal tumor incidence or burden were found between male Apc1638N offspring of different paternal diet groups. Although in female Apc1638N offspring there were no differences in tumor incidence or multiplicity, a stepwise increase in tumor volume with increasing paternal B vitamin intake was observed. Interestingly, female offspring of SUPP and DEF fathers had a significantly lower body weight than those of CTRL fed fathers. Moreover, hepatic trigylcerides and cholesterol were elevated 3-fold in adult female offspring of SUPP fathers. Weanling offspring of the same fathers displayed altered expression of several key lipid-metabolism genes. Hundreds of differentially methylated regions were identified in the paternal sperm in response to DEF and SUPP diets. Aside from a few genes including Igf2, there was a striking lack of overlap between these genes differentially methylated in sperm and differentially expressed in offspring.

Conclusions: In this animal model, modulation of paternal B vitamin intake prior to mating alters offspring weight gain, lipid metabolism and tumor growth in a sex-specific fashion. These results highlight the need to better define how paternal nutrition affects the health of offspring.

Fig 1. Design of animal experiment.

Apc1638N (+/−), mice heterozygous for truncation mutation in the Apc gene; WT (+/+), C57BL6/J mice wild-type for Apc gene; DEF, B-vitamin deficient; CTRL, B-vitamin replete; SUPP, B-vitamin supplemented; M, male; F, female.

Fig 2. Body weight of Apc^1638N offspring of fathers fed diets differing in B vitamin content.

A) Body weight of DEF compared to CTRL offspring, by sex. B) Body weight of SUPP compared to CTRL offspring, by sex. Repeated-measures ANOVA for paternal diet effect p = 0.64 and 0.02 in males and female offspring respectively. Paternal diets: DEF, B-vitamin deficient; CTRL, B-vitamin replete; SUPP, B-vitamin supplemented.* denotes significant difference (in females) compared to CTRL (p ≤0.05) by t-test. n = 14–22 per group.

Fig 3. Hepatic lipid concentrations of adult Apc^1638N offspring of fathers fed diets differing in B vitamin content.

A) Hepatic total cholesterol concentration. B) Hepatic triglyceride concentration. Paternal diets: DEF, B vitamin deficient; CTRL, B vitamin replete; SUPP, B vitamin supplemented. * denotes significant difference compared to CTRL of same sex (p<0.05). n = 13 per diet group.

Fig 4. Top networks of differentially expressed genes between CTRL and SUPP offspring unique to females.

A). Network associated with ‘Lipid Metabolism’, ‘Behavior’ and ‘Nervous System Development and Function’. Score = 36. B). Network associated with ‘Glutathione Depletion in Liver’, ‘Drug Metabolism’ and ‘Protein Synthesis’. Score = 44. Red = up-regulated in SUPP, Green = down-regulated in SUPP. Unshaded genes are present in the biological network but were not significantly altered in our dataset. The network score is a negative log p value of the Fisher exact test, which is testing whether these genes are grouped by chance.

Fig 5. Overlap between differentially methylated genes in sperm and differentially expressed genes in offspring.

A) CTRL v DEF comparison. Common in sperm and female offspring: Smc4, Spon2, Gpd2, Aurka, Tspan8, Tbc1d10a, Lss, Abhd2, Fdps, Hsd17b7, Dsg1c. Common in sperm and male offspring: Lrrc16a. Common in sperm and female and male offspring: Apol9b. B) CTR v SUPP comparison. Common in sperm and female offspring: 1300002K09Rik, Cyp3a16, Ethe1, Igf2, 1810011O10Rik. Common in sperm and male offspring: Col27a1.

Fig 6. Effect of paternal B vitamin supplementation on hepatic gene expression and triglyceride concentration in female offspring.

Gene expression levels are expressed as Fragments Per Kilobase Of Exon Per Million Fragments Mapped (FPKM). Triglycerides are expressed as μg/mg protein. CTRL, control; SUPP, supplemented (father’s diet).^a q<0.05, ^b p = 0.002 vs CTRL.