Detailed data on body weight gain and selected metabolite serum levels in the F0 founders are presented in ESM Table 5. The number of pups was similar in all groups; no effect of HFSSD or folate treatment on the number of pups was detectable.
Body weight and length
In the F1 generation, a paternal HFSSD diet resulted in body weight increase, which appeared to be more pronounced in female offspring (ESM Fig. 1). The male and female PatHFSSD/MatCD offspring were 8% and 6% heavier than their control littermates, respectively (p ˂ 0.05 in male offspring, p = 0.066 in female offspring). Folate intervention given to pregnant dams and folate food supplementation given to founders before mating both failed to reduce F1 progeny body weight.
At the end of the study, male and female PatHFSSD/MatCD offspring were 3.7% and 3.2% longer than their control littermates, respectively (p ˂ 0.01 for both sexes; Tables 1 and 2). In male and female offspring, folate given to pregnant dams decreased body length (by 3.0% and 2.7%, p < 0.05 and p < 0.01 vs PatHFSSD/MatCD, respectively). Folate given to founders before mating failed to reduce F1 progeny body length (Tables 1 and 2). The liver weight was increased in female PatHFSSD/MatCD offspring (p < 0.01 vs PatCD/MatCD) but not in the male offspring.
Feeding the F0 founders with HFSSD impaired the glucose homeostasis of their F1 offspring in a sex-specific manner. Female F1 progeny were more susceptible to glucose intolerance, as assessed by OGTT. When F1 female offspring of the PatHFSSD/MatCD group were 100 days old, serum glucose levels were elevated 60 min after glucose intake in the OGTT compared with levels in the PatCD/MatCD offspring (p < 0.001; Fig. 2b) and the AUC for glucose following the OGTT was also increased (p < 0.01 for PatHFSSD/MatCD vs PatCD/MatCD; Fig. 2d). In contrast to the F1 females, there were no significant alterations in glucose homeostasis in male F1 offspring except for serum glucose levels, which were elevated 30 min after glucose intake in the PatHFSSD/MatCD group compared with levels in the PatCD/MatCD offspring (p < 0.05; Fig. 2a, c).
In female F1 offspring, folate intervention given to pregnant dams (group) restored glucose tolerance (p < 0.05 for PatHFSSD/MatCD+F vs PatHFSSD/MatCD, 60 min after glucose intake, Fig. 2b) and significantly decreased glucose AUC following the OGTT (p < 0.01 for PatHFSSD/MatCD+F vs PatHFSSD/MatCD, Fig. 2d). Folate supplementation given to F0 founder male rats (PatHFSSD+F/MatCD) failed to restore glucose tolerance in their F1 offspring (Fig. 2a, b).
With the exception of lowered fasting glucose in PatHFSSD+F/MatCD vs PatHFSSD/MatCD male F1 offspring (p < 0.01), there were no significant differences in fasting glucose and insulin levels between the groups of male and female F1 offspring (Tables 1 and 2).
Male and female F1 offspring of HFSSD-fed F0 founder rats revealed mild hepatic steatosis (p < 0.01 and p < 0.05 vs PatCD/MatCD, respectively; Fig. 3a, b) manifesting as microvesicular steatosis (Fig. 3c, d). Minor hepatocyte degeneration was observed in all paternally programmed groups (data not shown). In PatHFSSD/MatCD offspring, the hepatic steatosis score was increased by 57% (p < 0.01) and 52% (p < 0.05) vs control male and female counterparts, respectively (Fig. 3a, b). Neither of the folate intervention groups showed any significant beneficial effects regarding steatosis.
In female F1 offspring born to male rats exposed to the HFSSD diet prior to mating liver fibrosis was increased (65% increase in periportal fibrosis score; p < 0.01 PatHFSSD/MatCD vs PatCD/MatCD; Fig. 4b). The increase in liver fibrosis in the female offspring was abolished by folate treatment of the dams during pregnancy (p < 0.01 PatHFSSD/MatCD+F vs PatHFSSD/MatCD, Fig. 4b). None of the study groups in the F1 male progeny showed any fibrotic changes around portal vessels. There were no statistical differences in parenchymal fibrosis levels and inflammation between any of the study groups in male offspring (Fig. 4a and Table 1). Liver triacylglycerol content did not significantly differ between the groups of F1 offspring, regardless of the sex (Tables 1 and 2).
Hepatic gene expression
We selected genes from studies showing that glucose tolerance in offspring was clearly differently regulated as a result of paternal high-fat diet during roughly two full rounds of spermatogenesis before mating [16, 29, 30]. Hepatic expression levels of Lcn2 mRNA were affected in both male and female F1 offspring of fathers fed HFSSD; folate treatment of parent rats normalised Lcn2 expression levels (Fig. 5b). Hepatic expression of Ppara mRNA was elevated in female F1 offspring only (1.51-fold, p ˂ 0.05) and was restored by folate treatment given to both dams and founders (p ˂ 0.05), whereas Ppara mRNA revealed no significant regulation in male F1 progeny (Fig. 5a). The analysis of hepatic mRNA expression of other genes (selected either based on a hypothesis-driven approach or based on micro-array data) revealed only minor differences between study groups in both male and female F1 progeny (Table 3).
To identify yet unknown genes that are differentially expressed in offspring of male rats given an unhealthy diet prior to mating, we performed a liver whole-genome array RNA sequencing approach. We chose the most promising candidate genes based on p values and fold change in the arrays and conducted real-time quantitative PCR (Table 3, Fig. 5a, b). The most prominent alterations were seen in the expression of Lcn2 and Tmcc2 genes which showed elevation in female offspring born to male rats exposed to an unhealthy diet (p ˂ 0.05, Fig. 5b and Table 3). Similar alterations in Lcn2 gene expression were seen in male offspring.
Pancreatic islets in the F1 offspring of both sexes born to male rats exposed to the HFSSD diet prior to mating had a decreased beta cell density (−20% in male and −15% in female F1 offspring, (p < 0.001 PatHFSSD/MatCD vs PatCD/MatCD; Fig. 6a, b). In female F1 progeny, folate given to either pregnant dams or F0 founders resulted in elevation of beta cell density when compared with their counterparts born to parents with no folate intervention (4.3% and 3.3% after folate supplementation given to dams and founders, respectively, p < 0.05 vs PatHFSSD/MatCD, Fig. 6b). However, neither of the parental folate interventions restored pancreatic beta cell density in the F1 male progeny (Fig. 6a). There were no significant differences in total number of islets, islet size distribution or islet area per mm2 pancreas section between the study groups of F1 offspring (Tables 1 and 2).
Pancreatic gene expression
Pancreatic candidate genes were selected as described for the liver, see above. The analysis of mRNA expression profiles of selected genes (Pparg, Ikbke, Ppara, Foxo1 and Fos) in the pancreas revealed only minor differences between study groups in both male and female progeny of the F1 generation (Table 4).
Global DNA methylation in the liver
Regardless of the F1 offspring sex, the rate of global DNA methylation in the liver was 1.52-fold elevated in PatHFSSD/MatCD offspring compared with their PatCD/MatCD control counterparts (p ˂ 0.05, Fig. 7a, b). In female F1 offspring, the treatment of the F0 dams with folate restored global methylation to a normal level (p ˂ 0.05 PatHFSSD+F/MatCD vs PatHFSSD/MatCD, Fig. 7b). However, effects of folate treatment of either the F0 founders or dams on the reduction in global DNA methylation rate was not statistically significant in the male offspring (Fig. 7a).
DNA methylation of specific target genes in the liver
The methylation rate of CpG islands in the promoter region of Ppara, Lcn2 and Tmcc2 genes was analysed. The differences among the groups were not significant, most likely due to the limited number of samples analysed (n = 5 per group) (ESM Table 6). We also investigated the potential correlation between the mRNA expression and the methylation rate of CpG islands in the promoter region of Ppara, Lcn2 and Tmcc2. Interestingly, the methylation rate of some CpG islands in the promoter region of Lcn2 showed significant negative correlation with Lcn2 mRNA expression in female offspring (ESM Table 7). Moreover, to investigate whether the methylation rate of a given CpG site is correlated with other CpG sites within the promoter region, Pearson correlation matrices were calculated and plotted as heat maps for each group (Fig. 5c, d and ESM Table 8). The resulting group-specific correlation patterns were clearly different. Regarding the correlation matrices of the methylation rate of CpG islands within the Ppara promoter, positive correlations, indicated by dark blue, were more predominant in the offspring born to fathers on a normal diet when compared with the offspring born to fathers on an unhealthy diet, even following folate treatment of both parents. On the other hand, regarding the correlation matrices of the methylation rate of CpG islands within the Lcn2 promoter, positive correlations were more predominant in the offspring born to fathers on an unhealthy diet when compared with the offspring born to fathers on a normal diet or those born to fathers fed an unhealthy diet where fathers or mothers had been treated with folate.
ANCOVA models considering litter size
As stated above, litter size was not significantly different between the groups. However, to investigate the influence and the potential bias of litter size, ANCOVA models, considering litter size as a covariate, were calculated for variables that were significantly different in the ANOVA analyses. Indeed, litter size was associated with several readouts, yet results overall were not affected by litter size (ESM Table 9).