Folic acid supplementation in a mouse model of diabetes in pregnancy alters insulin sensitivity in female mice and beta cell mass in offspring

Epidemiological studies have reported discrepant findings on the relationship between folic acid intake during pregnancy and risk for gestational diabetes mellitus (GDM). To begin to understand how folic acid impacts metabolic health during pregnancy, we determined the effects of excess folic acid supplementation (5× recommendation) on maternal and fetal offspring metabolic health. Using a mouse (female C57BL/6J) model of diet‐induced diabetes in pregnancy (western diet) and control mice, we show that folic acid supplementation improved insulin sensitivity in the female mice fed the western diet and worsened insulin sensitivity in control mice. We found no unmetabolized folic acid in liver from supplemented mice suggesting the metabolic effects of folic acid supplementation are not due to unmetabolized folic acid. Male fetal (gestational day 18.5) offspring from folic acid supplemented dams (western and control) had greater beta cell mass and density than those from unsupplemented dams; this was not observed in female offspring. Differential sex‐specific hepatic gene expression profiles were observed in the fetal offspring from supplemented dams but this differed between western and controls. Our findings suggest that folic acid supplementation affects insulin sensitivity in female mice, but is dependent on their metabolic phenotype and has sex‐specific effects on offspring pancreas and liver.


| INTRODUCTION
Gestational diabetes mellitus (GDM) is broadly characterized as any degree of glucose intolerance first detected during pregnancy. 1][4][5] Folate is critical for development and required for DNA synthesis, amino acid homeostasis, and the synthesis of the methyl donor, S-adenosylmethionine (SAM). 6Folic acid supplementation, the synthetic oxidized form of folate, is recommended for all women of child-bearing age for the prevention of neural tube defects (NTD). 7ndividuals at risk for an NTD-affected pregnancy, including those with type 1 or type 2 diabetes, obesity (BMI >30 kg/m 2 ), or a previous NTD-affected pregnancy are advised to take more folic acid (1-4 mg/day) than is recommended for a low risk pregnant individual (0.4 mg/day). 7urthermore, grain products in many countries, including Canada and the United States, are fortified with folic acid to ensure adequate folate intake during the early stages of pregnancy for prevention of NTDs. 8However, standard prenatal supplements often contain more than the recommended 0.4 mg of folic acid, [9][10][11] resulting in many women consuming above the upper tolerable intake level (1.0 mg/ day) 10,12,13 and raising blood folate concentrations. 14,15iscrepant findings on the relationships between folate intake before and during pregnancy and GDM have been reported.Findings from the Tongji Maternal and Child Health Cohort in China (n = 4353) indicated that women who consumed ≥0.8 mg/day folic acid supplement at least 4 weeks before pregnancy and continued for up to 16 weeks of pregnancy had a greater odds (adjusted odds ratio [95% CI]: 2.09 [1.30-3.36]) of developing GDM, based on a 75-g oral glucose tolerance test at 24-28 weeks of pregnancy, compared to women who took no folic acid supplement or consumed less than 0.4 mg/day folic acid supplement. 2 Similarly, the Shanghai Preconception cohort study (n = 1058) reported that folic acid supplementation in early pregnancy was associated with greater odds (adjusted odds ratio [95% CI]: 1.73 [1.19-2.53]) of GDM, based on 75 g oral glucose tolerance test at 24-28 weeks gestation. 4n contrast, findings from the Nurses' Health Study II in the United States (n = 14 553 women; n = 20 199 pregnancies) reported that women with adequate total folate intake (≥0.4 mg/day) from supplements and food before pregnancy had a lower relative risk (relative risk [95% CI]: 0.83 [0.72-0.95]) of GDM, based on self-reported physician diagnosis, compared to women who consumed <0.4 mg/day total folate before pregnancy. 3Moreover, women who took ≥0.6 mg/day folic acid supplement before pregnancy had a 30% lower relative risk of GDM compared to women who did not take a folic acid supplement before pregnancy. 3gnificant gaps remain in the understanding of the impact of folic acid intake and folate status on the metabolic health of women during pregnancy and whether it contributes to or prevents the pathophysiology of GDM.The prospective cohort studies 2-5 are difficult to interpret because of differences in the methods used for nutritional assessment, intake of other micronutrients, GDM diagnosis, and a lack of understanding on the effect of folic acid on metabolic health during pregnancy.Given the limitations and ethical considerations of conducting studies in pregnant individuals, the present study determined the effects of excess maternal folic acid supplementation (5× recommendations) in a mouse model of diet-induced diabetes in pregnancy (Western diet) on maternal and offspring (male and female) health at gestational day (E)18.5.We included assessments of the fetuses because of the evidence that excess folic acid supplementation during pregnancy also impacts the metabolic health of the offspring.We previously reported that maternal excess folic acid supplementation (5× recommendation) during pregnancy and lactation was associated with greater adiposity and glucose intolerance in adult female, but not male, C57BL/6J mouse offspring. 16,17

| Animals and diets
The study design is outlined in Figure 1.Female and male C57BL/6J mice were housed (2-4 mice/cage) under a standard 12-h light-dark cycle with ad libitum F I G U R E 1 Study design schematic: Female C57BL/6 mice (n = 11/14/group) were fed one of four diets for 13 weeks prior to breeding, and throughout gestation.At 11 weeks, ISTs were performed; at 12 weeks, intraperitoneal insulin tolerance tests (IPITTs) were performed; at 13 weeks, intraperitoneal glucose tolerance tests (IPGTTs) were performed.Tissue (maternal and fetal offspring) was collected at embryonic day 18.5.CD, control diet; CDF, control folic acid supplemented diet; WD, western diet; WDF, western folic acid supplemented diet.
access to food and water.The mice were gifted by Dr. Francis Lynn (Department of Surgery, UBC).At three weeks of age, female mice (n = 11-14/group) were weaned on to one of four diets (Research Diets Inc; New Brunswick, NJ, USA): control (10% kcal fat, 2 mg/ kg diet folic acid), control supplemented with folic acid (10% kcal fat, 10 mg/kg diet folic acid), western (45% kcal fat, 2 mg/kg diet folic acid), or western supplemented with folic acid (45% kcal fat, 10 mg/kg diet folic acid).Dams fed the western diet were used as a model of diet-induced diabetes in pregnancy.This models the condition of women with obesity and glucose intolerance/prediabetes prior to conception.The folic acid content of the unsupplemented control diet and western diet met AIN-93G diet recommendations. 18Further details of the diet composition are in Table S1.Dams were weighed weekly.Body composition was determined using quantitative magnetic resonance imaging (EchoMRI, Echo Medical Systems; Houston, TX, USA).After 13 weeks on diet, female mice were bred with age-matched male mice fed the control diet.Breeding was confirmed the morning after mating with a vaginal plug and assigned as E0.5.Female mice lacking vaginal plugs were returned to the breeding colony.On E18.5, dams were sacrificed, and maternal and fetal tissues were collected.

| Physiological assessment of glucose homeostasis
Before each assessment, female mice were fasted for five hours and a blood glucose measurement was obtained via glucometer (OneTouch Verio Meter, LifesScan; Malvern, PA, USA).At 11 weeks on diet, a glucose-stimulated insulin secretion test (IST) was performed: mice were injected with 0.75 g d-dextrose/kg lean mass (D9434, Sigma-Aldrich, MO) and saphenous blood was collected at 2, 15, and 30 min post-injection.Serum insulin was quantified by ELISA (80-INSMSU-E01, ALPCO, NY).At 12 weeks on diet, an intraperitoneal insulin tolerance test (IPITT) was performed: mice were injected with 0.75 U insulin/kg lean mass (Novolin®ge Toronto; Mississauga, Canada) and glucose concentrations were measured by tail prick at 15, 30, 60, 90, and 120 min post-injection.At 13 weeks on diet, an intraperitoneal glucose tolerance test (IPGTT) was performed: mice were injected with 0.75 g d-dextrose/kg lean mass and blood glucose was measured by tail prick at 15, 30, 60, 90, and 120 min post-injection.We did not conduct physiological assessments of glucose homeostasis during pregnancy to minimize stress on the dams and the developing offspring.

| Fetal sex determination
Genomic DNA was extracted from fetal tail snips using the Gentra Puregene Tissue Kit (158667, Qiagen, Toronto, Canada).Multiplex PCR was used to simultaneously amplify a 273 base pair sequence of the Sry gene and a 203 base pair sequence of Actb as the internal control (Table S2).

| Immunohistochemical analyses
For each fetal pup, 3 pancreatic sections separated by 30 μm each were stained for insulin and glucagon.Paraformaldehyde-fixed, paraffin-embedded pancreatic sections were stained for insulin and glucagon using a primary antibody cocktail of insulin (1:100, rabbit antimouse, 4950 Cell Signaling) and glucagon (1:500, mouse monoclonal anti-mouse, G2654, Sigma-Aldrich) and a secondary antibody cocktail against rabbit IgG (A-11012, Invitrogen) and mouse IgG (A28175, Invitrogen, Burlington, Canada).Images of whole pancreas and insulin and glucagon-positive cells were visualized and tiled with a BX61 fluorescence microscope (Olympus; Tokyo, Japan) and quantified by Fiji ImageJ software. 19Beta cell mass and alpha cell mass were calculated as the insulin-or glucagon-positive area expressed as a percentage of the whole pancreas area, respectively.Beta cell density was calculated as the number of insulin-positive cells per 100 mm 2 of whole pancreas area.

| Metabolite assessment
Liver samples were homogenized and protein concentration was determined by the Bradford Assay 20 with the Quick Start™ Bradford 1X Dye Reagent (5000205, Bio-Rad, Hercules, CA).Total lipids were extracted from homogenized liver using a modified method of Folch et al. 21followed by colorimetric quantification of triglycerides (Triglyceride Reagent Set, 23-666-412, Pointe Scientific, MI).
Water-soluble choline metabolites (free choline, phosphocholine, glycerophosphocholine, and betaine) were quantified by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) using stable-isotope labeled internal standards in the Analytical Core for Metabolomics and Nutrition at the B.C. Children's Hospital Research Institute.Hepatic S-SAM and S-adenosylhomocysteine (SAH) were quantified by high-performance liquid chromatography with UV detection using the method of Fell et al. 22 with modifications by Miller et al. 23 Serum concentrations of 5-methyltetrahydrofolate (5-MTHF) were determined by liquid chromatographytandem mass spectrometry (LC-MS/MS) as previously described. 24Briefly, samples were prepared by adding 5 volumes of methanol containing 0.1% formic acid and internal standard ( 13 C 5 -5-MTHF) to 25 μL of serum.Following centrifugation, clear extracts were injected into a Shiamdzu Nexera LC system coupled to a Sciex 5500 QTrap mass spectrometer.Folate in liver tissue was determined by LC-MS/MS as previously described. 25Samples were prepared to measure monoglutamate forms of folate intermediates following a deconjugation protocol. 25Filtered sample extracts were injected into an Acquity UPLC system coupled to a Xevo TQ MS spectrometer (Waters Corporation, Milford, MA, USA).

| Offspring hepatic gene expression
RNA was extracted from fetal livers using the AllPrep Mini Kit (Qiagen).RNA (1 μg) was reverse transcribed to cDNA libraries using the NEBNext Ultra II directional RNA library prep kit with poly(A) enrichment module (New England Biolabs (E7760S, E7490S, MA, USA) and quantified by the Qubit HS DNA Assay (Q32851, Invitrogen) and Bioanalyzer High Sensitivity DNA Chip (5067-4626, Agilent, CA, USA).Libraries were pooled and sequenced on the NextSeq 500 platform (Illumina; San Diego, CA).Sequences were aligned to the Gencode vM25 transcriptome (mm10) using STAR version 2.7.3a 26 and transcripts were quantified using Salmon version 1.4.0. 27Differential expression was determined using DESeq 2 v1.24 28 with filtering parameters of adjusted p value <.1 and Log2 fold change greater than or equal to 1. Unsupervised hierarchical clustering was conducted using pheatmap.Differential expression and clustering analyses were performed in R version 3. The functional enrichment analysis was performed in gProfiler version e105_eg52_p16_e84549f with g:SCS multiple testing correction method applying a significance threshold of .05. 29

| Statistical analyses
The Shapiro-Wilk test and Levene's test were used to check the normality and homogeneity of variance of the data, respectively.Data that were not normally distributed were natural log transformed.Two-way analysis of variance (ANOVA) was used to determine the effect of maternal diet and folic acid supplementation.If a significant interaction (p < .05)was observed, a subsequent t-test was performed to determine the effect of folic acid supplementation separately in the control diet and western diet groups.Repeated measures 2-way ANOVA was used where appropriate (growth curve, IST, IPITT, and IPGTT data).Data from one male and one female offspring per litter were used for analyses; data in male and female offspring were analyzed separately.Analyses were performed using SPSS (version 27, IBM; Armonk, NY, USA).Individual data points are presented; bar graphs represent mean ± standard deviation (SD).p-Values for differential gene expression were calculated with the Wald test with Benjamini-Hochberg adjustment and gene set enrichment in differentially expressed genes with Fisher's exact test with Benjamini-Hochberg adjustment.

| Folic acid supplementation increases body weight and fat mass and alters insulin tolerance tests in female mice
Dams fed the western diet gained more body weight from weeks 1-13 on the diet and had greater fat mass at 13 weeks than control dams (Figure 2A,B).Interestingly, control and western dams supplemented with folic acid gained more body weight and fat mass than unsupplemented dams (Figure 2A,B).At E18.5, western dams had larger subcutaneous and gonadal fat pads than control dams; there was no effect of folic acid supplementation on body fat distribution in the dams at E18.5 (Figure 2C,D).
Glucose homeostasis was assessed in the dams just prior to breeding.Western dams had higher fasting blood glucose and serum insulin, and impaired glucose tolerance (Figure 2E-G).Interestingly, changes in blood glucose concentrations during the IPITT were impacted by folic acid supplementation, but the effect was dependent on the diet.Dams fed the control diet supplemented with folic acid had a quicker rise in blood glucose concentrations during the IPITT, suggesting reduced insulin sensitivity, similar to the unsupplemented western dams (Figure 2H).In contrast, dams fed the western diet supplemented with folic acid had a similar response in blood glucose concentrations during the IPITT as observed in the unsupplemented control dams suggesting improved insulin sensitivity.We observed no effect of supplementation on beta-cell function in the dams; western dams had greater insulin secretion in response to glucose compared to control dams (Figure 2I).Western dams had lower pancreas weights (Figure 2J), but no effect of diet or supplementation on beta-cell mass was observed (Figure 2K).

| Folic acid supplementation increases folate status to a greater extent in diabetes during pregnancy but has no effect on liver folate stores
As expected, maternal folic acid supplementation increased serum 5-methyltetrahydrofolate (5-MTHF), the predominant circulating form of folate, in control and western dams (Figure 3A,B).However, the western dams had higher serum 5-MTHF than control dams, despite the same folic acid content of the diets (Figure 3A).The differences in circulating 5-MTHF were not reflected in liver total folate stores (Figure 3C).Folic acid supplementation in control and western dams had no effect on liver total folate or individual folate forms (Figure 3C-G); unmetabolized folic acid was not detected in liver.We further observed no effect of folic acid supplementation on maternal liver SAM and SAH concentrations (Figure 3H-J).Western dams had lower liver SAM and SAH concentrations than control dams (Figure 3H,I).

| Maternal folic acid supplementation has sex-specific effects on fetal offspring body weight and beta-cell density
Fetal female offspring from western dams had lower body weight than offspring from control dams (Figure 4A).No differences in body weight were observed in male offspring (Figure 4B).Greater beta cell mass and beta cell density were observed in male offspring, but not female offspring, from control and western dams supplemented with folic acid (Figure 4C-F).No impact of maternal diabetes (western diet) on offspring beta cell mass or density was observed.We observed no effects of maternal diet or folic acid supplementation on offspring pancreas weight and alpha cell mass (Figure 4G-J).

| Maternal folic acid supplementation and diabetes during pregnancy impact fetal offspring liver one carbon metabolism
Fetal female offspring from control and western dams supplemented with folic acid had higher liver SAM concentrations and SAM/SAH ratios compared to fetal female offspring from unsupplemented dams; no effect of maternal folic acid supplementation on liver SAH was observed (Figure 5A-C).Maternal diabetes had no effect on liver SAM, SAH, or SAM/SAH ratios in fetal female offspring (Figure 5A-C).The impact of maternal folic acid supplementation on liver SAM and SAH was dependent on maternal diet in fetal male offspring.Lower liver SAH concentrations were observed in fetal male offspring from western dams supplemented with folic acid compared to those from unsupplemented western dams (Figure 5E), whereas liver SAH concentrations were slightly higher in fetal male offspring from control dams supplemented with folic acid compared to those from unsupplemented control dams (Figure 5E).No impact of maternal folic acid supplementation or diabetes on liver SAM or SAM/SAH ratios in fetal male offspring were observed (Figure 5D,F).Fetal male and female offspring from western dams had smaller livers compared to those from control dams (Figure 5G,H).
Given the evidence that maternal obesity and diabetes can impact fatty liver disease in the offspring, 30 we further investigated whether maternal folic acid supplementation impacts liver triglyceride concentrations and water-soluble choline metabolites in the fetal offspring.Liver triglyceride concentrations were lower in fetal female offspring from western dams compared to control dams; no effect of folic acid supplementation was observed (Figure 6A).No effect of maternal folic acid supplementation or diabetes on liver triglyceride concentrations was observed in fetal male offspring (Figure 6B).Free choline and betaine concentrations were higher in liver from fetal male offspring from western dams compared to those from control dams; this was not observed in female offspring (Figure 6C-F).Maternal diet and folic acid supplementation had no effect on liver glycerophosphocholine or phosphocholine in female or male offspring (Figure 6G-J).
We further assessed the impact of diabetes and maternal folic acid supplementation on fetal offspring hepatic gene expression.Offspring from folic acid supplemented control dams had differential expression of Gprc5a, Krt7, Apoa5, Xlr3b, and Gm45140 in males and Fabp2, Apoa4, Prap1, and Dysf in females, respectively (Figure 7A,B).Unsupervised hierarchical clustering of differentially expressed genes revealed perfect separation based on maternal folic acid supplementation status for male, but not female offspring (Figure 7A,B).No Gene Ontology terms were enriched in the downregulated genes in males (Gprc5a and Krt7) but one term was enriched in the upregulated genes: Gene Ontology: biological process (GO:BP), "positive regulation of very-low-density lipoprotein particle remodeling."Twelve terms were enriched in the four genes upregulated in female offspring, primarily related to lipid transport, binding, digestion, and absorption (Figure 7C).
Compared to control dams, folic acid supplementation in western dams had a greater impact on offspring liver gene expression.We found 29 genes with upregulated expression and 5 genes that were downregulated in male offspring from western dams supplemented with folic acid compared to male offspring from unsupplemented western dams, with perfect separation based on maternal folic acid supplementation (Figure 7D).No differences were observed in female offspring.Nine terms were enriched in the upregulated genes, pathways predominantly related to protein binding, disaggregase activity, and microbubble nucleation (Figure 7E).

| DISCUSSION
][4][5] We report three main findings.First, folic acid supplementation 5× recommendations affected insulin sensitivity in the dams, but the effect was dependent on the diet of the dam.Folic acid supplementation improved insulin sensitivity in the dams fed the western diet, to levels observed in the unsupplemented control dams; whereas in control dams, it worsened insulin sensitivity, to levels observed in the unsupplemented western dams.Second, it is unlikely that the effect of folic acid supplementation on insulin sensitivity in the dams is due to differences in liver folate metabolism.Dams supplemented with folic acid had higher serum 5-MTHF concentrations but we found no differences in liver folate concentrations and no unmetabolized folic acid in liver.Third, maternal folic acid supplementation impacted fetal male offspring pancreas and liver, but had negligible effects on fetal female offspring.We observed greater beta cell mass and beta cell density and differential liver gene expression profiles in fetal male offspring, but not in fetal female offspring.

F I G U R E 4
Maternal folic acid supplementation increases fetal male offspring beta-cell density at gestational day 18.5: Body weight of fetal female offspring (A) and male offspring (B) at gestational day 18.5.Beta-cell mass in fetal female offspring (C) and male offspring (D).Beta cell density in fetal female offspring (E) and male offspring (F).Alpha-cell mass in fetal female offspring (G) and male offspring (H).Pancreas weight in fetal female offspring (I) and male offspring (J).Beta-and alpha-cell mass were calculated as the insulin-or glucagonpositive area expressed as a percentage of the whole pancreas area, respectively; beta-cell density was calculated as the number of insulinpositive particles per 100 mm 2 of the whole pancreas area.Statistical significance was determined by a 2-way ANOVA.Data are presented as mean ± standard deviation.
diet-induced mouse model of diabetes in pregnancy that we used in our study models the condition of an individual with obesity and glucose intolerance/ prediabetes prior to conceiving.We confirmed this phenotype in the dams prior to breeding and verified that western diet-fed dams had glucose intolerance, elevated fasting blood glucose and serum insulin concentrations, and greater adiposity relative to control dams.Most interestingly, maternal folic acid supplementation impacted insulin tolerance, implying some improvement in insulin sensitivity of the dams fed the western diet.Our findings suggest that excess folic acid supplementation is beneficial under conditions of metabolic stress as it pertains to insulin sensitivity.However, we observed no impact of excess folic acid supplementation on glucose tolerance in the dams.We conducted the physiological assessments of glucose homeostasis just prior to breeding to avoid causing unnecessary stress by conducting the assessments during pregnancy.This contrasts how GDM is diagnosed in pregnant individuals, which usually occurs at 24-26 weeks of pregnancy in low-risk individuals and at 20 weeks gestation in highrisk individuals. 1he underlying mechanisms of how folic acid supplementation improves insulin sensitivity in dams fed the western diet are not known.We analyzed the IPITT curves by repeated measures ANOVA as recommended, 31 rather than investigating differences at each individual time point.The glucose excursion curves for the control dams supplemented with folic acid and the western dams began to rise and deviate from the other groups at 30 min post-insulin injection, suggesting reduced insulin sensitivity.However, it should be noted that the greatest differences between the groups emerged in the later part of the IPITT, suggesting the group differences may also involve differences in counter regulatory responses (catecholamines, cortisol, glucagon) to hypoglycemia. 32Estimates suggest that in male mice, counter regulatory mechanisms are stimulated once blood glucose concentrations fall below 4.4 mmol/L. 33In our female mice, the mean blood glucose values for the groups at any of the time points during the IPITT did not fall below 4.4 mmol/L.However, this could reflect sex-specific differences in counter regulatory responses to hypoglycemia.
We postulated that the differences observed during the IPITT may involve direct effects of folic acid supplementation on folate metabolism in the liver.Surprisingly, we found no unmetabolized folic acid in liver from the dams, which contrasts other reports that found unmetabolized folic acid in liver of nonpregnant female and male mice. 34,35Rodents have higher dihydrofolate reductase activity than humans, 36 augmenting their capacity to metabolize folic acid, which could explain why we did not detect unmetabolized folic acid in the liver of the supplemented dams.However, despite observing higher serum 5-MTHF concentrations in supplemented control and western dams, we found no significant differences in liver total folate concentrations.It is possible that folate concentrations in other tissues such as skeletal muscle, kidney, and pancreas are more reflective of serum folate concentrations.Cellular uptake of folates is regulated by different transport proteins, including the reduced folate carrier (SLC19A1) and the proton-coupled folate transporter (SLC46A1), that have different affinities for folate forms and vary in expression across different cell types.It has also been suggested that folic acid itself may inhibit the folate cycle given that to be metabolically active, it is reduced to dihydrofolate then tetrahydrofolate by dihydrofolate reductase before its conversion to 5-MTHF by methylenetetrahydrofolate reductase (MTHFR) (Figure 3B).Christensen et al. reported lower 5-MTHF concentrations, but no differences in total folate concentrations, in liver from male BALBc mice fed a 10× folic acid supplemented diet for 6 months and lower MTHFR activity in liver extracts incubated with excess folic acid. 34Furthermore, folic acid uptake is inhibited in high folate conditions (100 μmol/L) in intestinal and kidney cell lines (Caco-2 and HK-2 cells, respectively). 37bservational studies have reported positive relationships between high folate status during pregnancy with GDM or other indicators of insulin resistance and F I G U R E 7 Maternal diabetes and folic acid supplementation alter fetal offspring liver gene expression patterns: Heatmaps showing unsupervised hierarchical clustering of gene expression patterns affected by folic acid supplementation in liver from (A) fetal male offspring, and (B) fetal female offspring from control dams (CDF vs. CD).Functional enrichment visualizations of (C) genes upregulated in liver from fetal female offspring (CDF vs. CD).Gene expression patterns affected by folic acid supplementation in liver from (D) fetal male offspring from western dams (WDF vs. WD) (adjusted p-value <.1, log2 fold change >1, n = 5/group).The colors show the degree of intensity of log 2 normalized read counts, with yellow indicating higher and blue indicating lower expression and (E) genes upregulated in liver from fetal male offspring (WDF vs. WD).Gene ratio was calculated as intersection size/term size and enrichment was performed using a significance threshold of <.5 in gProfiler.9][40] Surprisingly, the improved insulin sensitivity in the western dams supplemented with folic acid was accompanied by higher serum 5-MTHF concentrations compared to supplemented control dams.The reason for these differences is not known but may reflect differential effects of the diets on the intestinal absorption of folic acid, the balance between renal reabsorption and urinary excretion of folates, and/or other differences in liver folate metabolism.
Little is known about the impact of excess folic acid supplementation in GDM on offspring health.][43][44] We reported that adult female, but not male, offspring from control dams supplemented with folic acid during pregnancy/lactation have glucose intolerance. 17In the current study, we observed greater beta-cell density and mass in male fetal offspring from both control and western dams, suggesting excess folic acid supplementation has some impact on fetal beta-cell development independent of maternal metabolic health.However, maternal diabetes independently modified the gene-regulatory effect of folic acid in liver from the offspring, with the gene signature induced by folic acid supplementation in control dams distinct from western offspring.6][47] Others have also reported effects of parental folic acid supplementation on adult offspring beta cells.The reduced beta-cell density observed in adult female rat offspring from fathers fed a high fat, sucrose, and salt diet was rescued if the dams were supplemented with folic acid. 48Conversely, lower fasting serum insulin concentrations and beta cell mass were reported in adult male and female offspring of dams supplemented with much higher folic acid levels (40 mg/kg diet) C57BL/6 dams. 43here is a paucity of data on mechanisms underlying the impact of folic acid on beta-cell function and/or insulin production.However, some recent in vitro studies suggest folate may play and important role in insulin production.Impaired insulin synthesis was observed in pancreatic RINm5F beta-cells cultured in folic acid-deficient media, which could not be rescued by replenishment of folic acid. 49Furthermore, folic acid promoted proliferation and upregulated insulin secretion in high glucose conditions in porcine pancreatic stem cells. 50Folinic acid, the stable form of formyltetrahdyrofolate, was shown to increase beta-cell differentiation in zebra fish and cultured neonatal porcine islets. 51n summary, we report that maternal folic acid supplementation interacts with diet to alter insulin sensitivity in dams and has sex-specific effects on beta cell density and mass and hepatic gene expression in fetal offspring.Further investigations are warranted to elucidate the role of folic acid on insulin production.Moreover, it will be salient to identify candidate epigenetic regulators that may be facilitating the folic acid-driven changes in offspring hepatic gene expression to further apprehend these effects.

F I G U R E 2
Folic acid supplementation increased body weight and fat mass and affects insulin sensitivity in female mice: (A) Body weight and (B) % fat mass at 13 weeks on diet.Weight of (C) gonadal and (D) subcutaneous fat pads at gestational day 18.5.(E) Fasting blood glucose; (F) serum insulin; (G) intraperitoneal glucose tolerance test; (H) intraperitoneal insulin tolerance test; and (I) IST before breeding.(J) Pancreas weight and (K) beta-cell mass at gestational day 18.5.Statistical significance was determined by a 2-way ANOVA.Data as presented as mean ± standard deviation.

F I G U R E 5
Sex-specific effects of maternal diabetes and folic acid supplementation on fetal offspring liver SAM and SAH: (A) S-adenosylmethionine (SAM), (B) S-adenosylhomocysteine (SAH), and (C) SAM/SAH ratio in liver from fetal female offspring.(D) SAM, (E) SAH, and (F) SAM/SAH ratio in liver from fetal male offspring.(G) Female and (H) male liver weight.Statistical significance was determined by a 2way ANOVA.Data are presented as mean ± standard deviation.

F I G U R E 6
Maternal diabetes has sex-specific effects on fetal offspring liver triglycerides and choline metabolites: (A) Female and (B) male liver triglycerides, (C) female and (D) male free choline (FC); (E) female and (F) male betaine; (G) female and (H) male glycerophosphocholine (GPC); and (I) female and (J) male phosphocholine (PChol).Statistical significance was determined by a 2-way ANOVA.Data are presented as mean ± standard deviation.