Essential nutrient supplementation prevents heritable metabolic disease in multigenerational intrauterine growth-restricted rats

Abstract

Intrauterine growth restriction (IUGR) confers heritable alterations in DNA methylation, rendering risk of adult metabolic syndrome (MetS). Because CpG methylation is coupled to intake of essential nutrients along the one-carbon pathway, we reasoned that essential nutrient supplementation (ENS) may abrogate IUGR-conferred multigenerational MetS. Pregnant Sprague-Dawley rats underwent bilateral uterine artery ligation causing IUGR in F1. Among the F2 generation, IUGR lineage rats were underweight at birth (6.7 vs. 8.0 g, P < 0.0001) and obese by adulthood (p160: 613 vs. 510 g; P < 0.0001). Dual energy X-ray absorptiometry studies revealed increased central fat mass (Δ+40 g), accompanied by dyslipidemic (>30% elevated, P < 0.05) serum triglycerides (139 mg/dl), very-LDLs (27.8 mg/dl), and fatty acids (632 µM). Hyperglycemic-euglycemic clamp studies and glucose tolerance testing revealed insulin resistance. Conversely, IUGR lineage ENS-fed rats did not manifest MetS, with significantly lower body weight (p160: 410 g), >5-fold less central fat mass, normal hepatic glucose efflux, and >70% reduced circulating triglycerides and very-LDLs compared with IUGR control-fed F2 offspring (P < 0.01). Moreover, increased methylation of the IGF-1 P2 transcriptional start site among IUGR lineage F2 offspring was reversed in ENS (P < 0.04). This is an initial demonstration that supplementation along the one-carbon pathway abrogates adult morbidity and associated epigenomic modifications of IGF-1 in a rodent model of multigenerational MetS.—Goodspeed, D., Seferovic, M. D., Holland, W., Mcknight, R. A., Summers, S. A., Branch, D. W., Lane, R. H., Aagaard, K. M. Essential nutrient supplementation prevents heritable metabolic disease in multigenerational intrauterine growth-restricted rats.

The gestational environment is a key arbiter of the relative risk of health and disease in later life. In utero metabolic substrate restriction and uteroplacental insufficiency leading to intrauterine growth restriction (IUGR) is thought to bestow lifelong epigenetic changes that are maladaptive to the postnatal environment. This phenomenon is referred to as the developmental origins of health and disease hypothesis and was originally identified by associating low birth weight neonates with increased mortality from coronary heart disease much later in life (1, 2). IUGR has now been associated with increased incidence of health conditions including vascular dysfunction (3, 4), type II diabetes and dyslipidemia (5, 6), obesity, and hypertension (2, 7, 8). A symptomatic grouping of these IUGR health complications later in life is broadly characterized as metabolic syndrome (MetS), a condition approaching global epidemic prevalence (9). The clinical criteria for MetS is evolving as it struggles to account for large differences in global populations, age, and race (10). Currently, the consensus of clinical criteria includes the following: dyslipidemia, characterized by elevated triglycerides, ApoB lipoproteins, and reduced HDL; elevated blood pressure; and altered glucose homeostasis, with elevated central fat mass/obesity and insulin resistance (10–12).

Maternal influences, including poor nutrition and uteroplacental insufficiency, are associated with IUGR and adult MetS (13–16). Rodent IUGR models that restrict either protein or carbohydrates have demonstrated adult-onset MetS associated with important epigenetic regulatory changes to key metabolic pathways (17–20), and our previous data in primates have demonstrated that a caloric-dense maternal diet can influence epigenetic changes in the fetal liver (21). Although nutrient deprivation is a risk for MetS regardless of IUGR etiology (13, 22–24), maternal deprivation is not a predominant cause of IUGR in all but the most underdeveloped countries. Rather, uteroplacental insufficiency is the predominant cause of fetal nutrient restriction leading to IUGR, which is independent of maternal nutrition. Thus, maternal nutrient restrictive models inadequately replicate the broad deprivation of metabolites and essential nutrients (including methyl donors) in placental insufficiency, as well as other important changes such as hypoxia and acidosis, waste product accumulation, and associated biochemical changes such as inflammation, key regulatory factor alterations, and broad transcriptional changes particularly associated with hypoxia. Ergo, the surgical bilateral uterine artery ligation procedure represents a more appropriate model to study epigenetic changes associated with placental insufficiency as it replicates restriction due to circulatory defects and in this way leads to reduced maternal/fetal exchange. The immediate consequences are persistent in utero hypoinsulinemia, hypoglycemia, acidosis, and hypoxia (25, 26).

Extensive study of animal models has uncovered some specific epigenetic-mediated molecular mechanisms implicated in long-term heath complications (20, 27–35). IGF-1 is the primary driver of late gestational and postnatal growth and has been implicated in altered weight and development (36, 37). Decreased expression is associated with insulin resistance later in life, as well as increased incidence of MetS (38). In a number of model systems, a perturbed in utero environment lending epigenomic modifications (both histone variations and differential methylation) leads to IGF-1 expression changes (33–35, 39). Of interest to our studies herein, an identical bilateral uterine artery ligation procedure results in altered IGF-1 expression changes, which are accompanied by site-specific differential methylation (33). Of the splice variants, the IGF-1B was shown to be particularly variable both in its expression and its methylation (33).

Although DNA methylation is relatively stable, there are developmental time points, such as gamete imprinting, preimplantation development, and post-midgestational development, when methylation changes are active (40). These windows provide intervention points where methylation is particularly plastic and, for imprinting and postimplantation events, amenable to dietary influence. Dietary methylation supplementation is now known to lead to variable gene expression (41, 42). These epigenetic changes associated with methylation supplementation continue to influence biology after weaning (43). For example, glucose tolerance in later life is associated with methyl deficiency during gestation (44), and supplementation of methyl donors alters hepatic promoter methylation and the transcriptional profile in nonalcoholic fatty liver disease (45).

DNA methylation changes as causative agents of disease have long been established. It was discovered that methylation of our DNA is sufficiently sensitive to essential dietary methyl sources and cofactors to cause human disease over a decade ago (46). For example, methyl sources related to seasonal food consumption in Gambia affect DNA methylation patterns (47). This sensitivity is likely caused by methyl group substrates and cofactors that constitute the one-carbon metabolism pathway, many components of which are essential, and therefore can only be derived from dietary sources (48). Methylation of CpG islands is catalyzed by DNA methyltransferases with S-adenosyl methionine as the methyl donor, synthesized from methionine via dietary methyl donors or one-carbon metabolism (49). Notably, the essential amino acid methionine is a methyl donor, itself dependent on B₁₂ as a methionine synthase cofactor, which also uses a downstream product of folic acid as a substrate. In an alternate liver pathway, methionine is synthesized from betaine, which is derived from choline. Further, several of the enzymes are zinc dependent (46, 50). Methylation is further influenced along the renal arginine-dependent nitric oxide pathway (51).

Studies have made use of these essential nutrients to study how epigenetic methylation changes relate to disease. For example, male mice have shown epigenetic reprogramming of sperm that leads to birth defects in offspring when fed a folate-deficient diet (52). In another example, rats using essential nutrient methyl-donor dietary supplementation (ENS) have shown modifications in DNA methylation and decreased hepatic triglyceride accumulation and nonalcoholic fatty liver disease, which are one of the first hepatic alterations of metabolic syndrome (53). In humans, methylation changes to IGF-1 that were associated with nonalcoholic fatty liver disease were partially reversed as a result of nutritional adjustment following bariatric surgery (54).

Given that DNA methylation is modifiable by ENS of the diet, we hypothesized that supplementation initiated after weaning and continued through gestation and lactation of the F1 generation would abrogate the heritable adult metabolic disease resulting from in utero IUGR among F2 offspring. To study these effects, we rendered uteroplacental insufficiency-induced IUGR by performing late-gestation bilateral uterine artery ligation on grandparental (P1) pregnant Sprague-Dawley rats at e19, with subsequent delivery of the F1 pups at e21. The F1 generation was allocated onto either control or ENS-supplemented diet at weaning (p21) and then allowed to breed spontaneously; the F1 generation was not surgically manipulated during pregnancy. Regardless, the resultant IUGR lineage rats in the F2 generation weighed significantly less at birth than the sham lineage pups. By p160, a sex-specific MetS phenotype was apparent. IUGR lineage males fed a control diet exhibited obesity, increased central fat mass accumulation, glucose intolerance, insulin resistance, and increased triglyceride, very-LDL (VLDL), and fatty acids. ENS-fed IUGR lineage males exhibited no obesogenicity, glucose intolerance, insulin resistance or increases in triglycerides, VLDL, or fatty acids, indicating a diet induced reversal of metabolic disease. Accompanying these notable and significant phenotypic alterations are significant site-specific differences in DNA methylation of the IGF-1 promoter 2 region. Specifically, we demonstrate that there is an observable and significant corresponding increase in methylation among F2 generation IUGR lineage rats relative to sham controls; a finding that reversed with ENS diet supplementation in the F1 and F2 generation. Collectively, we observe a multigenerational transmission of the IUGR phenotype with manifestation of adult MetS and variations in site specific methylation of the P2 promoter of IGF-1, which is abrogated by dietary supplementation of one-carbon intermediates in the F1 generation.

MATERIALS AND METHODS

IUGR rat model development

The experiments were conducted with the review and approval of the University of Utah Animal Care committee. Surgical procedures were performed as previously described (55). Briefly, pregnant Sprague-Dawley rats were obtained on e17 and acclimated to their environment for 2 d prior to surgery. On day e19, pregnant females were anesthetized with an intraperitoneal injection of xylazine (8 mg/kg) and ketamine (40 mg/ml). After confirmation of anesthetization, bilateral uterine artery ligation was performed to produce the uteroplacental insufficiency IUGR model, or sham surgeries were performed for controls. Pups were born via cesarean section after 21 d of gestation, and litters were culled to 8 pups. On p21, suckling F1 offspring were weaned onto an ad libitum lifelong diet of either control rat chow (Harlan Teklad 8640; Harlan Laboratories, Indianapolis, IN, USA) or a custom-formulated isocaloric Harlan Teklad 8640 rat chow supplemented with 15 mg of folic acid, 15 mg of choline, 1.5 mg of B₁₂, 15 g of betaine, 11 g of l-methionine, 50 g of l-arginine, and 150 mg of zinc for every kilogram of Harlan Teklad 8640, constituting the ENS diet. Mating pairs were established by p80, prior to the development of F1 MetS. Mating pairs were sham^mat × sham^pat, IUGR^mat × sham^pat, sham^mat × IUGR^pat, and IUGR^mat × IUGR^pat for both the control diet and ENS diet, giving a total of 8 different mating sets. Four hours after spontaneous delivery of the F2 generation (N = 512), fetal weights were obtained, and litters were again culled to 8 pups (N = 32). On p21, the F2 generation was weaned onto the same diet of their maternal and paternal lineage. Rats were weighed both at birth and at p160.

Dual energy X-ray absorptiometry

Dual energy X-ray absorptiometry (DEXA) Norland XR-36 Bone Densitometer (Norland Products, Inc., Cranbury Township, NJ, USA) was used to assess rat whole body global and regional body composition. DEXA relies on X-ray technology to separate the lean and fat mass based on density and has been previously used extensively for nutritional studies (56, 57). Rats were anesthetized before measurements with an intraperitoneal injection of xylazine (8 mg/kg) and ketamine (40 mg/ml) and then placed in prone position with extremities extended and stabilized with tape to enable full scanning of the radius and ulna. Spatial resolution was 6.0 × 6.0 mm. Scans were performed at a speed of 60 mm/s. The scan field width was 12 cm, and scanning time for each animal was <3 min. Following the scan, rats were immediately euthanized. To determine central fat mass, a region of interest including the torso area was extracted from the whole body scan, and fat mass was determined.

Fasting serum lipid profiling

Adult p160 rats were fasted for 12 h, and then blood was collected by nicking the end of the tail with a scalpel once. Blood flow was maintained throughout by gentle stroking of the tail. Bleeding was stopped after sampling by dipping the tail in alcohol and subsequent application of direct pressure. A coronary risk and lipid profiling panel was run on each serum sample. Triglyceride and VLDL concentrations were obtained by clinical laboratory testing performed by ARUP Laboratories (Salt Lake City, UT, USA) using standard quantitative enzymatic assays. Free fatty acids were measured from serum using the Roche Half-Micro Test Kit (Indianapolis, IN, USA). The kit was scaled down for use in a 96-well plate (one-fifth suggested amount for each reagent; 10 µl serum). All preparation was performed according to the manufacturer’s instructions. Palmitate standard was made by dissolving 74 mg palmitate into a solution of 35 ml ethanol and 65 ml 6% Triton X-100 to make a high standard of 2.878 mM and then serially diluted. Absorbance was assessed on a UV/Vis microplate reader. A standard curve was generated from which total fatty acid contents from serum samples were interpolated using the average of triplicate wells.

Glucose tolerance test

Adult p160 rats were fasted for 6 h and then weighed. Blood samples were collected by tail vein as previously described. A sample of blood was collected before administration of glucose, which was then administered orally to each rat (2.5 g glucose/kg body weight). Blood samples were subsequently collected at 15, 30, 45, 60, 90, and 120 min after glucose challenge from the tail as described. Serum insulin levels were measured using an ELISA kit.

Hyperinsulinemic-euglycemic clamp studies

The use of hyperinsulinemic-euglycemic clamp studies was carried out to determine insulin resistance, as the most accurate and sensitive laboratory method that is specific to hepatic insulin resistance (58). Experiments were performed as described previously (59). Adult p160 rats were fasted for 6 h prior to commencement of the experiment. Following aseptic placement of jugular (for infusion) and carotid (for blood sampling) catheters, animals were allowed to recover to presurgical weight (7–10 d). Clamps were performed in conscious unrestrained animals using swivels and tethers (Instech, Plymouth Meeting, PA, USA) to allow uninterrupted movement of the animal without disruption of infusion lines. Hyperinsulinemia was initiated by intravenous infusion of insulin (4 mU/kg per min). Blood was sampled from arterial lines in 7 min intervals and analyzed with a Beckman Glucose analyzer II (Beckman Coulter, Fullerton, CA, USA). Euglycemia was maintained by variable infusion of 20% dextrose. Steady-state glucose (∼130 mg/dl) was achieved ∼90 min after initiating hyperinsulinemia and maintained for ≥45 min. Glucose infusion rates were calculated as the average glucose infusion rate during the steady-state period. Additional blood samples were taken before initiating hyperinsulinemia and at the end of the clamp for analysis of insulin. [¹⁴C]Glucose and [³H]2-deoxyglucose were given as a bolus during the steady state for estimation of hepatic glucose output and 2-deoxyglucose uptake, as previously described (60).

Methylation-specific PCR

To map IGF-1 promoter methylation changes, a standard PCR-based method following bisulfite modification was performed as described previously (33). Briefly, primer sets were designed to probe 3 regions of CpG sites of the IGF-1 P2 transcription start site. These were forward 5′-GGAGGGTTTAATTTATAAAAGATTTTAG, reverse 5′-CCCAAACCACTTCCTTACCTAA; forward 5′-GTTGTTGTTGTTATTGTTYGTGGTA, reverse 5′-CTAAAATCTTTTATAAATTAAACCCTCC; and forward 5′-TTAGGTAAGGAAGTGGTTTGGG, reverse 5′-CACTATCTCAACTACCAAACCAC. PCR conditions were as follows: 95°C for 10 min, and then 94°C for 30 s, annealing at 53°C for 30 s, and 72°C for 30 s for 35 cycles. Primers were designed and figures prepared with the use of University of California, Santa Cruz Genome Browser (61). A subset of 8 p21 animals was used for each condition. PCR products were cloned into the vector pSC-A (Stratagene, Cedar Creek, TX, USA), and 6 colonies from each cloning were inoculated on SeqPrep 96 plates (Edge Biosystems, Gaithersburg, MD, USA). The SeqPrep 96 Plasmid Prep Kit (Edge Biosystems) was used to prepare the plasmid DNA according to manufacturer’s instructions, and the DNA was then sequenced.

Statistical analyses

Statistical analyses were performed using Prism v6.0 (GraphPad Software Incorporated, La Jolla, CA, USA). One-way ANOVA with Bonferroni’s post hoc analysis was used to compare mean changes in weight. Student’s t test was used to compare body mass composition and serum lipid profile and mean hepatic glucose output (HGO). Means are reported ± sem. Significance was determined at P < 0.05.

RESULTS

Development of a multigenerational heritable IUGR rat model

P1 Sprague-Dawley female rats fed a control diet underwent either a sham or bilateral uterine artery ligation surgery at day 19 after gestation (Fig. 1A). The rats spontaneously delivered either the F1 sham control or IUGR lineage litters (Fig. 1A) and were thereafter culled (n = 8 pups per litter). On p21, the F1 offspring were weaned onto isocaloric diets of either control or ENS consisting of the control diet with added folic acid, choline, B₁₂, betaine, methionine, arginine, and zinc. To establish the F2 line, F1 rats were mated at approximately day 80 of life (Fig. 1B). IUGR females and males were either fed control or ENS diets and were paired to establish the IUGR^mat/IUGR^pat lineage groups. Rats with either maternal or paternal IUGR lineage were also mated with sham partners of the same diet. Additionally, sham males and females were paired and fed either control or ENS diets. The F1 dams were fed their lifelong control or ENS diet throughout gestation and suckling. At weaning, the F2 rats were placed on the same diet as their F1 parents. All experiments were performed on the F2 generation.

Figure 1.

Mating scheme for heritable growth restriction in rats. A) Pregnant female Sprague-Dawley rats (P1) underwent either a bilateral uterine artery ligation or sham surgery at gestation day e19. Uterine ligation F1 rats exhibiting IUGR or sham control rats were then assigned to a lifelong diet of either control chow (control) or ENS chow at weaning (p21). B) At P80, males and females were mated in all combinations of both maternal and paternal IUGR lineage with and without ENS. The resultant F2 generation was assigned the parental diet of either ENS or control at p21, generating 8 F2 study groups (n = 512 animals).

Multigenerational heritable growth restriction modifiable by ENS in F1 and F2

After spontaneous delivery, fetal weights were obtained and litters were culled (n = 8 pups per litter). Pups derived from the Sham^mat/Sham^pat lineage were significantly larger at birth than the pups derived from the IUGR^mat/IUGR^pat lineage (Fig. 2A, B). Quantification of birth weights revealed IUGR^mat/IUGR^pat pups from the maternal control diet to be 16% less at birth than the Sham^mat/Sham^pat lineage (P < 0.0001; 6.7 vs. 8.0 g, respectively). Pups from either IUGR^mat/Sham^pat (5.8 g) or Sham^mat/IUGR^pat (6.1 g) lineage also weighed 27% and 24% less at birth, respectively, than the Sham^mat/Sham^pat lineage (P < 0.0001). With maternal ENS-fed rats, a 30% decrease in birth weight with the IUGR^mat/IUGR^pat lineage was observed, which was statistically identical to the nonsupplemented IUGR^mat/IUGR^pat lineage rats. Similarly, Sham^mat/IUGR^pat ENS-treated rats showed a marked decrease in birth weight (35%, P < 0.0001), whereas IUGR^mat/Sham^pat ENS-treated rats did not show any significant decrease in weight compared with Sham^mat/Sham^pat controls (Fig. 2B). These results suggest a multigenerational effect of low birth weight for the F2 pups, resultant from uteroplacental insufficiency of the F1 generation.

Figure 2.

Uterine artery ligation in rats confers heritable growth restriction and adult obesity. A) F2 pups at birth from F1 IUGR^pat/IUGR^mat lineage and F1 Sham^pat/Sham^mat lineage show substantial birth weight differences. B) Differences in mean birth weight of the F2 generation from the F1 lineage fed either a control or ENS diet. Significant differences from Sham^pat/Sham^mat control diet are indicated. C) F2 males at p160 from the IUGR^pat/IUGR^mat lineage fed either a control or ENS diet. Mean p160 weights for F2 male (D) and female (E) rats, bred from the F1 lineage that were fed either a control or ENS diet. Significant differences from Sham^pat/Sham^mat control diet are indicated. (^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001, 1-way ANOVA with Bonferroni’s post hoc test).

Male and female pups reassessed at p160 demonstrated a significant effect of ENS treatment. Figure 2C shows the IUGR^mat/IUGR^pat males fed an ENS diet were smaller than the IUGR^mat/IUGR^pat males fed a control diet, which was true for females as well. Males derived from IUGR^mat/IUGR^pat fed a control diet weighed ∼17% greater than males from the Sham^mat/Sham^pat (Fig. 2D; P < 0.0001). Similarly, the females from the IUGR^mat/IUGR^pat fed a control diet weighed ∼19% more than the Sham^mat/Sham^pat females (Fig. 2E; P < 0.0001). Interestingly however, both male and female IUGR^mat/IUGR^pat rats reported an average ∼ 20% reduction in weight gain when fed an ENS diet compared with the Sham^mat/Sham^pat control diet (Fig. 2D, E; P < 0.0001). For IUGR^mat/Sham^pat rats fed a control diet, there was no statistically significant difference in weights from the Sham^mat/Sham^pat and only a small 8% decrease for Sham^mat/IUGR^pat, in female rats only (P < 0.05; Fig. 2D, E). However, when fed an ENS diet, the IUGR^mat/Sham^pat males and females weighed significantly less than the Sham^mat/Sham^pat (P < 0.0001 and 0.01, respectively), but Sham^mat/IUGR^pat was significantly decreased for males only (P < 0.0001; Fig. 2D, E). Whereas the Sham^mat/Sham^pat showed no significant change in mean weight when fed an ENS diet, the obesity evident in the IUGR^mat/IUGR^pat control fed was abolished by ENS, with a more pronounced effect apparent in the males.

Multigenerational heritable obesity and dyslipidemia modifiable by ENS in F1 and F2

To determine the basis for the weight changes in IUGR, the relative body fat and lipid profile of the animals was characterized as an indication of metabolic health. IUGR^mat/IUGR^pat rats fed a control diet were observed to have visibly more fat mass than either IUGR^mat/IUGR^pat rats fed an ENS diet or Sham^mat/Sham^pat rats (Fig. 3A). Lean mass and central fat mass were quantified on adult animals (p160) using DEXA on a clinical caliber Norland scanner, for which representative scans are shown in Fig. 3B. No significant difference in lean mass in either males or females was apparent (Fig. 3C, D). The IUGR^mat/IUGR^pat males fed a control diet, however, had 3 times more central fat mass than Sham^mat/Sham^pat control fed (P = 0.05), which was also significantly greater than either IUGR^mat/IUGR^pat and Sham^mat/Sham^pat males fed the ENS diet (P = 0.01; Fig. 3E). IUGR^mat/IUGR^pat females fed a control diet also had significantly more central fat mass than the IUGR^mat/IUGR^pat females fed an ENS diet (Fig. 3F; P < 0.01). The increased central fat mass observed in IUGR^mat/IUGR^pat males and females suggests that ENS prevents central fat deposition.

Figure 3.

ENS prevents adult obesity. A) Representative image of the central fat found in IUGR males fed either a control or ENS diet. B–F) DEXA scans for body fat analysis based on tissue density. B) Representative DEXA scans of Sham^pat/Sham^mat and IUGR^pat/IUGR^mat males fed either a control or ENS diet. Mean lean mass in F2 males (C) and females (D) fed either a control or ENS diet are indicated compared with Sham^pat/Sham^mat control. Differences in mean central fat mass of Sham^pat/Sham^mat and IUGR^pat/IUGR^mat F2 males (E) and females (F) fed either a control or ENS diet compared with IUGR^mat/IUGR^pat control fed. (^aP < 0.05 considered significant, independent Student t test).

Fasting serum lipid profiles were then assessed as markers of dyslipidemia, which accompanies atherosclerotic disease and is an indication of MetS. Triglycerides were elevated in IUGR^mat/IUGR^pat control fed males and females (P < 0.04), with a significant reduction in both Sham^mat/Sham^pat and the IUGR^mat/IUGR^pat ENS fed for both males and females (P < 0.003; Fig. 4A, B). Similarly, VLDL appeared elevated for both males and females. Although it did not reach statistical significance (P = 0.13 and 0.09, respectively), VLDL was nevertheless lowered to normal levels with ENS supplementation for both sexes (P < 0.005; Fig. 4C, D). Serum free fatty acid levels also exhibited the trend of lineage-induced elevation in IUGR^mat/IUGR^pat control diet, with males showing ∼50% elevation (P = 0.009), which was reversed in both IUGR^mat/IUGR^pat and Sham^mat/Sham^pat ENS-fed rats (P < 0.01; Fig. 4E, F). The alterations in serum lipid concentrations suggest that ENS supplementation reverses the IUGR-induced increases that characterize metabolic disease.

Figure 4.

ENS prevents adult dyslipidemia. Mean triglyceride levels in F2 males (A) and females (B) fed either a control or ENS diet compared with IUGR^mat/IUGR^pat control fed. Mean VLDL levels in F2 males (C) and females (D) fed either a control or ENS diet compared with IUGR^mat/IUGR^pat control fed. Mean free fatty acid levels in F2 males (E) and females (F) fed either a control or ENS diet compared with IUGR^mat/IUGR^pat control fed. (P < 0.05 considered significant and all significant values annotated, independent Student t test).

Multigenerational heritable glucose intolerance and dysglycemia modifiable by ENS in F1 and F2

Given the strong relationship between insulin resistance and MetS, hyperinsulinemic-euglycemic clamp studies with carbon 14-labeled glucose and tridium-labeled 2-deoxy glucose tracers were performed on adult animals. These clamp studies were used to discern the hepatic insulin resistance, as opposed to overall or peripheral, as a highly specific indication of metabolic dysregulation. HGO was significantly higher in IUGR^mat/IUGR^pat males than Sham^mat/Sham^pat males fed a control diet (Fig. 5A; P < 0.001), indicative of impaired hepatic insulin sensitivity. When fed an ENS diet, IUGR^mat/IUGR^pat males exhibited enhanced insulin-mediated suppression of HGO, reversing them to Sham^mat/Sham^pat control levels (Fig. 5A; P < 0.02). Similarly significantly distinct HGO profiles were observed among female offspring (Fig. 5B).

Figure 5.

ENS prevents adult insulin resistance. Mean HGO of Sham^pat/Sham^mat and IUGR^pat/IUGR^mat F2 males (A) and females (B) fed either a control or ENS diet compared with IUGR^pat/IUGR^mat control. Representative insulin response of Sham^pat/Sham^mat and IUGR^pat/IUGR^mat F2 male and female without (C, D) and with (E, F) ENS supplementation, respectively, after glucose challenge. Insulin was measured by ELISA (P < 0.05 considered significant and all significant values annotated, independent Student t test).

To test for insulin responsiveness, glucose tolerance tests were also performed. Serum insulin levels were increased in the IUGR^mat/IUGR^pat rats fed a control diet compared with Sham^mat/Sham^pat following a glucose challenge (Fig. 5C, D). At 50 min, the insulin levels of both male and female IUGR^mat/IUGR^pat rats failed to return to control baseline, and the levels were still severalfold elevated at 90 min compared with sham (Fig. 5C, D). For both male and female rats, their ENS-fed insulin response was very different. For males, insulin was not discernibly elevated in IUGR^mat/IUGR^pat rats fed the ENS diet compared with Sham^mat/Sham^pat (Fig. 5E). For females, there appeared to be a significant amelioration of the insulin response for ENS-fed IUGR^mat/IUGR^pat rats compared with control diet; however, insulin remained elevated above baseline at 50 min (Fig. 5F). Overall, the changes associated with IUGR and also the abrogation of the phenotype are considerably more pronounced in the male rats compared with the females.

IGF-1 promoter methylation in IUGR abrogated by ENS

To test whether ENS was acting to affect methylation of critical metabolic regulatory genes, we mapped the methylation status of the second promoter (P2) of the IGF-1 from liver tissue of the variously treated groups. The promoter map is depicted in Fig. 6A, where vertical lines are indicative of individual CpG dinucleotides, and amplicons from each of the areas I–III of the P2 promoter are shown. Sequencing of bisulfite-modified hepatic genomic DNA revealed site (CpG dinucleotide) specific differential methylation. Specifically, among IUGR^mat/IUGR^pat control-fed F2 offspring destined to manifest adult MetS, we observed significantly increased P2 methylation compared with Sham^mat/Sham^pat control-fed F2 offspring (P = 0.03). Consistent with our phenotypic analysis, there was no apparent effect of ENS alone on the non-IUGR lineage animals (Sham^mat/Sham^pat ENS fed; Fig. 6B, C). Promoter area II amplicons retained CpG sites where significant methylation changes occurred, whereas areas I and III showed minimal changes (Fig. 6B). When fed a diet supplemented with essential nutrients inclusive of the one-carbon intermediates, methylation of the P2 promoter CpG dinucleotides were significantly decreased in the IUGR^mat/IUGR^pat rats compared with control-fed offspring (P = 0.04; Fig. 6C). The altered methylation patterning in a site (CpG dinucleotide and region-specific manner) suggests that IUGR-mediated methylation changes in a critical metabolic organ are neither global nor indiscriminate. Further, the changes in promoter methylation appear in agreement with IGF-mediated restricted growth in F1 as previously reported (33).

Figure 6.

IGF-1 promoter methylation mediated by IUGR and ENS. A) The 3 analyzed regions of promoter 2 of IGF-1 are depicted together with the amplified areas sequenced. CpG dinucleotides are represented as vertical lines. In total, among the 3 indicated regions, the methylation status of 11 CpG dinucleotides was determined in bisulfite-treated hepatic tissue. B) Methylation patterns of CpG islands on the P2 transcription start site. Open circles are unmethylated CpGs, whereas closed circles indicate methylated. Promoter site in indicated on the horizontal axis. C) Quantitation of overall methylation changes at the 11 CpG sites for IUGR and Sham linage mice with and without supplementation. Differences from IUGR^mat/IUGR^pat are indicated (independent Student t test, sem).

DISCUSSION

In this study, we used our well-established bilateral uterine artery ligation model of uteroplacental insufficiency to demonstrate that IUGR induces MetS in the F1 generation (33, 34) and in the F2 generation. Our novel findings are consistent with other findings of multigenerational effects in the human population and a single prior maternal lineage rodent study (15, 62). Remarkably, the adult F2 generation exhibited excessive weight with increased central adiposity and a highly transformed metabolism. Additionally, the adult F2 phenotype included dyslipidemia, significant insulin resistance, and altered glucose metabolism, which altogether are characteristics of MetS. The observed changes were significant in the males, who exhibited much greater heritability compared with females. Most interestingly, the phenotypes were nearly completely reversed with ENS along the one-carbon metabolic pathway regardless of sex or amplitude. IGF-1 methylation was also shown to be reversed by ENS. These results suggest that adaptive epigenomic changes are modifiable and sufficiently plastic to reverse or prevent an inherited diseased epigenome.

The phenotypic consequences of bilateral intrauterine artery ligation to the F1 generation rats are well established and include the MetS phenotype of insulin resistance, diabetes, hypertriglyceridemia, dyslipidemia, and hypertension (13, 55, 62, 63). Additionally, a myriad of physiologic and biochemical changes underlying the phenotype have been found. These include metabolic changes related to altered islet development and dysglycemia (20), increased hepatic glucocorticoid activity (27), altered GLUT1 and GLUT2 expression (28), hepatic glucose production, hepatic one-carbon metabolism, altered hepatic fatty acid metabolism, muscle mitochondrial lipid metabolism, and cerebral chromatin structure (29–31, 64). There is evidence of compromised kidney development and function and an altered intestinal morphology (32, 65, 66). Further, important molecules related to development and to a MetS phenotype, such as IGF-1, PGC-1, CPTI, and PDX1, have been shown to be epigenetically altered (33, 34, 67, 68).

In these extensive and multigenerational experiments, we were able to enrich our deep phenotyping data across both the F1 and F2 generations with initial interrogations into key regulatory genes, such as IGF-1 (33, 34, 67, 68). We were guided in our decision of which genes and promoters to interrogate by the established biology, as well as the known role of methylation in regulating alternate splice sites for transcription of the IGF-1 P2 promoter (33). We show that CpG site-specific methylation of the IGF-1 P2 promoter was altered specifically and significantly in the liver with inclusion of one-carbon metabolism intermediates as dietary supplements. Of note, these modifications occurred only among animals arising from the IUGR lineage and thus accompany reversal of the adult MetS phenotype. Although at first glance it might be expected that ENS would lead to diffuse methylation effects in multiple organ and tissue types, it is particularly significant that IGF-1 (a central anabolic and developmental regulator with systemic endocrine function) is altered by ENS; this is consistent with prior findings in a single generation model of uterine artery ligation in the rat (33).

Hepatic expression of IGF-1 has diverse and systemic roles in development, growth, and glucose homeostasis, and it is known to be adaptively regulated in IUGR (69–71). Increased promoter methylation in the F1 IUGR rat liver dampens IGF-1 expression in what is also thought to be an additional epigenetically mediated adaptive mechanism (33). It is well established that the IGF-1 gene sequence has variable epigenetic regulation, and accompanying transcriptional changes have been shown repeatedly and by multiple investigators (33, 35, 39, 54). Despite this, the mechanisms for multigenerational inheritance observed here are thus far unclear. Aside from its role in growth and development, low levels of IGF-1, like those seen accompanying increased methylation following placental insufficiency (33), is critical to later glucose homeostasis and insulin resistance (37, 72, 73). Our findings herein significantly contribute to the role of epigenomic modifications in both regulating IUGR-mediated adult MetS and an accompanying variation in IGF-1 expression and demonstrate a favorable impact of one-carbon intermediate supplementation.

Although the metabolic effects to those born of IUGR pregnancies are well documented, fewer studies have reported on the multigenerational metabolic effects of IUGR. Recently, a study focusing on the F2 generation uncovered that rats with low maternal birth weight due to late-gestation (e19) uteroplacental insufficiency exhibited a sex-specific reduced insulin response and altered pancreatic morphology (55). In another study, female F2 rats exhibited increased hepatic weight with aberrant glucose and insulin metabolism (74). Natural experiments and comparisons to human populations have borne out the validity of the rodent IUGR models, finding similarities in epigenetic metabolic regulation (23, 33, 55, 65). Of particular interest, the Dutch famine studies have identified transgenerational epigenetic modifications and a propensity for increased adiposity and poor health in F2 as a result of F1 in utero metabolic restriction (23, 24). Therefore, although some aspects of metabolic dysfunction have been demonstrated, it was previously unclear that MetS can be passed down from F1.

The heritable MetS found in our IUGR model of uteroplacental insufficiency indicates there are sex-specific phenotypes for heritability. For instance, males are more severely affected than females in our model, which is highlighted by IUGR^mat/IUGR^pat lineage males developing the entire spectrum of MetS by adulthood. However, we are able to ameliorate the metabolic syndrome apparent in the IUGR^mat/IUGR^pat lineage male rats with feeding of an ENS diet. Conversely, although IUGR^mat/IUGR^pat lineage females show a trend toward MetS, the distinction between IUGR^mat/IUGR^pat compared with Sham^mat/Sham^pat was most evident with respect to adult weight. Similarly, the metabolic end points of insulin dyslipidemia and insulin resistance were considerably more robust for males. Nevertheless, there was both evidence of significant multigenerational inheritance of the adult MetS phenotype in IUGR lineage offspring, as well as a statistical amelioration when fed the ENS diet, regardless of sex. These results are consistent with reports from others that show sex-specific differences in IUGR (27, 62, 75–77). A suggested mechanism to account for the protected phenotype in females is thought to be through estrogen signaling, where estrogen regulates levels of choline, a critical downstream substrate of methionine synthesis. Higher levels of choline have been shown to be protective of metabolic deficits, potentially decreasing the effects of heritable epigenetic aberrations in females (78, 79).

We find the effectiveness of ENS supplementation remarkable in nearly reversing IUGR-associated adult metabolic morbidities. We intentionally initiated the ENS diet intervention in the F1 at the time of weaning and prior to the onset of adult MetS, in an effort to both introduce supplementation prior to adolescence, as well as prior to evidence of metabolic dysfunction. We demonstrated the development of the multigenerational phenotype and the adult MetS phenotypic reversal (but not multigenerational IUGR per se) is abrogated with an early in life ENS intervention.

Although the findings here present an interesting model of the principle of nongenomic coded inheritance and its implications for human health, there are nevertheless limitations. In humans, as in mice, a variety of factors, including a nonstandard environment, variable parental care, and unique nonepigenetically determined biology of the mother, can influence the development of the fetus and confer health altering changes that are not genetically heritable (80). Likewise, it must also be considered that the F2 germ line was exposed to relative restriction and rescuing ENS supplementation, whereas their F1 mothers were undergoing growth restriction in utero. There are examples of germ-line inherited changes that disappear in F3 (81). It is conceivable that the F2 effects and reversal thereof came about through epigenetic modifications in meiosis. Nevertheless, this does not diminish the significance that epigenetically programmed changes regardless of when they occurred are subject to intervention and mitigation with appropriate diet. It has been suggested that supplementation along the one-carbon pathway alters the biochemical balance of reactions for carbohydrate metabolism. As with most experiments of this kind, it is not possible to fully isolate the desired readouts resultant from genetic regulatory changes from the direct effects of treatment on metabolism. Although it is suggested here that maternal supplementation of ENS diet may stave off later metabolic disease, this conclusion is tempered by the use of an animal model. Further, our model of uteroplacental insufficiency is a particularly severe one.

To our knowledge, we are the first group to show a near-complete reversal of heritable MetS through dietary supplementation, as well as a multigenerational transmission down both maternal and paternal lineages. Using robust metrics of central fat mass, glucose tolerance, hepatic glucose efflux, serum lipid, and free fatty acids, we show that supplementation with essential nutrients along the one-carbon pathway prevents adult obesity, dyslipidemia, and insulin resistance, indicative of metabolic disease. The novel demonstration of a paternal inheritance of adult MetS has substantial translational implications, as it underlines the role of paternal, as well as maternal, factors as important to noncoded heritability of MetS. Identifications of the mechanisms and genetic targets of heritable epigenetic-mediated metabolic changes and the molecular mechanisms of phenotypic reversal through ENS supplementation will reveal insights into the causes of the developmental origins of health and disease as they relate to IUGR. Supplementation of IUGR offspring, as first identified at birth, may be a viable intervention to mitigate the risks conferred by multigenerational inheritance of metabolic-related diseases.

Glossary

DEXA

dual energy X-ray absorptiometry

ENS

essential nutrient supplementation

HGO

hepatic glucose output

IUGR

intrauterine growth restriction

MetS

metabolic syndrome

VLDL

very-LDL