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1Division of Neonatology and Developmental Biology, Department of Pediatrics, David Geffen School of Medicine at the University of California Los Angeles (UCLA), Los Angeles; and 2Harbor-UCLA Medical Center, Torrance, California
Submitted 1 September 2006 ; accepted in final form 12 December 2006
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ABSTRACT |
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 TOP ABSTRACT RESEARCH DESIGN AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, and PKC enzyme activity, with no change in total SHP2 and PTP1B concentrations in IUGR F2 compared with F2 CON. We conclude that transgenerational presence of aberrant glucose/insulin metabolism and skeletal muscle insulin signaling of the adult F2 IUGR female offspring is independent of the immediate intrauterine environment, supporting nutritionally induced heritable mechanisms contributing to the epidemic of type 2 diabetes mellitus. glucose transporter; metabolic imprinting; epigenetic inheritance
-cells in type 2 diabetes mellitus as well, an aberration that is passed on transgenerationally (3, 17, 27). Although mutations of genetic loci responsible for insulin production are inherited (33, 34), emerging information suggests epigenetic regulation underlying this transgenerational inheritance pattern (7, 13, 18).
In the first-generation (F1) adult female IUGR offspring with pre- and postnatal nutrient restriction, metabolic adaptations concerning glucose/insulin homeostasis consist of a diminution in glucose-induced insulin response with emerging hepatic insulin resistance (8) and aberrant skeletal muscle insulin signaling, culminating in insulin resistance of glucose transporter (GLUT)4 translocation (21, 32). Investigations (38) employing selective protein restriction during gestation and lactation in the F0 generation led to hypoinsulinemia in the F1 and second-generation (F2) female offspring with hyperglycemia in the latter, supporting inheritance of this phenotype. In a separate study (4), the development of gestational diabetes mellitus in the F1 adult IUGR offspring caused glucose intolerance and insulin resistance in the progeny. This phenotype of the F2 generation was a result of aberrant insulin secretion and diminished skeletal muscle GLUT4 expression (4). Although changes in the male and female F2 offspring may show subtle differences (38), the transgenerational persistence of aberrant glucose metabolism may be due to fetal adaptations that occur in response to a metabolically adverse F1 intrauterine environment. Alternatively, gestational diabetes in the F1 mother results in glucose toxicity targeted at fetal pancreatic -islets, thereby setting the stage for the subsequent development of glucose intolerance in the F2 offspring (4, 19, 20).
To distinguish between the differing mechanisms underlying the transgenerational persistence of metabolic perturbations in the IUGR, we hypothesized that inheritance plays a significant role independent of the immediate intrauterine metabolic environment of the F1 mother. We tested this hypothesis using embryo transfer methodology in which the progeny of an IUGR female rat was implanted into a metabolically controlled intrauterine environment. Persistence of dysregulated glucose/insulin homeostasis and aberrations in skeletal muscle insulin signaling in the F2 adult female offspring of an F1 IUGR mother that gestated and was postnatally groomed in a control nutritional/metabolic environment supports a role for epigenetic regulation in transgenerational metabolic programming.
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RESEARCH DESIGN AND METHODS |
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TOPABSTRACTRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Sprague-Dawley rats (7–8 wk old, 200–250 g; Charles River Laboratories, Hollister, CA) were housed in individual cages, exposed to 12:12-h light-dark cycles at 21–23°C, and allowed ad libitum access to standard rat chow. The National Institutes of Health guidelines were followed as approved by the Animal Research Committee of the University of California, Los Angeles.
Maternal Nutrient Restriction Model of IUGR
Pregnant rats (F0) received 50% of their daily food intake (11 g/day) beginning from day 11 through day 21 of gestation, causing nutrient restriction during late gestation and resulting in IUGR. The control counterparts received ad libitum access to rat chow (8). Both groups had ad libitum access to drinking water through gestation.
Postnatal Animal Maintenance (F1)
At birth, the litter size was culled to six. Newborn rats born to nutrient-restricted mothers continued to be reared by nutrient-restricted mothers that receive 50% of daily food intake through lactation (20 g/day; IUGR). The control (CON) pups born to control mothers were reared by control mothers with ad libitum access to rat chow (8).
Experimental Groups
Two groups of F1 donor animals were created, with 1) control pups reared by control mothers (CON) and 2) IUGR pups reared by nutrient-restricted mothers (IUGR) (Fig. 1). On day 21, the pups from two groups were weaned from the mothers and maintained as two animals per cage on a similar diet of standard rat chow until 2 mo of age.
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The 2-mo-old F1 CON and F1 IUGR female donor animals received 40 µg ip of gonadotropin-releasing hormone (Sigma L4513) and were mated with CON males on the fourth evening. Simultaneous mating was performed between recipient CON females and vasectomized males, and both pregnancy and pseudopregnancy were confirmed by the presence of a vaginal plug the following morning.
Collection of Eggs
Under inhalational 2.5% isoflurane anesthesia, the abdominal area of donor females from two experimental groups was shaved and scrubbed with betadine alcohol. A longitudinal incision (2 cm) was made taking sterile precautions to expose the horns of the uterus, oviducts, and ovaries. Oviducts and the uterus were removed and transferred at room temperature into a 35-mm petri dish containing M2 medium (M2-MR-051-F, M2 with hyaluronidase, specialty medium; Chemicon International, Temecula, CA). The oviduct was cut open, and eggs surrounded by cumulus cells were flushed out into M2 medium with hyaluronidase (M2 with hyaluronidase, specialty medium; Chemicon International). Fertilized oocytes were collected and maintained in M16 (5.55 mM glucose containing specialty medium; Chemicon International) microdrop cultures at 37°C for <2 h (range 1–2 h) until embryo transfer was performed.
Embryo Transfer Procedure
The recipient pseudopregnant rats were anesthetized by receiving intraperitoneal ketamine HCl (50 mg/kg), acepromazine maleate (1 mg/kg), and xylazine (4.8 mg/kg). Eye care was provided by the use of a bland ophthalmic ointment during the procedure to prevent corneal drying. Under a thermoneutral environment, using an isothermal pad, the lower paraspinal region was shaved and scrubbed three times, alternating with betadine and alcohol. Using sterile precautions, a 1-cm-long transverse incision was made to the left of the vertebral column at the level of the last thoracic rib. The oviduct was exposed and the infundibulum steadied with blunt forceps while 12-h-old pooled embryos (30–40 embryos) were transferred into the oviduct by inserting the transfer pipette into the ampular opening. The muscle and subcutaneous tissues were approximated with absorbable sutures, and the incision was closed with wound clips that were removed after 7–10 days. Animals were monitored closely until full recovery from anesthesia and surgery.
Postnatal Care of Embryo-Transferred Animals
Pregnant recipient females were allowed to deliver. The average litter size was comprised of 12 ± 2 pups. Each litter size was culled to six and was reared by control mothers to maintain consistent postnatal nutrition. Postweaning, the offspring had ad libitum access to standard laboratory rat chow.
Intravenous Glucose Tolerance Test
Fifteen-month-old awake adult female animals received 1 g/kg body wt of a 1:1 mixture of [2-2H]- and [6,6–2H2]glucose (>98% pure; Cambridge Isotope Laboratories, Andover, MA) via surgically placed jugular venous catheters (8). Serial blood samples (500 µl) were obtained for assessment of hormones, glucose concentrations, and isotopomer enrichment.
Insulin Tolerance Test
Fifteen-month-old awake adult female animals received 0.75 U/kg of human insulin via the jugular venous catheter, and blood was subsequently obtained at 0, 15, 30, and 60 min to measure glucose concentrations (32).
Body and Organ Weights
After body weight was measured the animals were anesthetized by inhalation of isoflurane, and organs/tissues (brain, heart, liver, kidneys, white adipose tissue, and brown adipose tissue) were removed and weighed individually.
Plasma Assays
Plasma glucose was measured by the glucose oxidase method (sensitivity = 0.1 mM; Sigma Diagnostics, St. Louis, MO). Insulin and leptin were quantified by enzyme-linked immunoabsorbent assays using rat standards and anti-rat insulin or leptin antibodies (sensitivity: insulin = 0.2 ng/ml, leptin = 0.04 ng/ml; Linco Research, St. Charles, MO). Corticosterone was quantified using a radioimmunoassay and an anti-rat corticosterone antibody (sensitivity 
5.7 ng/ml with apparent concentration at 95% B/Bo, Coat-A-Count; Diagnostic Products, Los Angeles, CA) (8, 32).
Gas Chromatography-Mass Spectrometry Analysis
Glucose was analyzed by gas chromatography-mass spectrometry using a modified method described by Szafranek et al. (28). All isotopomeric determinations were performed using a Hewlett-Packard gas chromatograph (model 6890) connected to a Mass Selective Detector (model 5973A; Hewlett-Packard, Palo Alto, CA). Electron impact ionization was used to characterize glucose positional isotopomers of [6,6-2H2]glucose at mass-to-charge (m/z) ratio 187 for C3–C6 and of [2-2H]glucose at m/z 242 for C1–C4 fragments (8).
Analysis and Interpretation of Glucose Tolerance Test
Mass isotopomer distribution was determined using the method of Lee et al. (15). The disappearance of the two isotopes, [2-2H]- and [6,6-2H2]glucose, was determined as the M1 label for [2-2H]glucose and the M2 label for [6,6-2H2]glucose (8). The difference between the disappearance rates of M1 and M2 was used as a measure of futile cycling (i.e., glucose to glucose 6-phosphate and back) (14).
Glucose Transporter Protein Analysis
Skeletal muscle preparation. Homogenates and subcellular fractions were prepared from 15-mo-old F2 adult female animals, as previously described (31). Plasma membrane (PM)-enriched subfractions were isolated as previously described, and the relative purity was determined (31). The homogenate and PM subfractions were stored at –70°C until Western blot analysis was undertaken.
Western blot analysis. The homogenates and the fractionated sarcolemma/PM samples were sonicated (60 sonic, Dismembrator; Fisher Scientific, Pittsburgh, PA) using two 50-s cycles of 5–7 W. The resulting suspension was centrifuged at 10,000 g at 4°C for 10 min and the supernatant subjected to Western blot analysis, as previously described (31). The affinity-purified rabbit anti-rat GLUT4 antibody (at 1:2,500 dilution) was used as the primary antibody. Glucose transporter protein concentrations were assessed by quantification of the protein bands by densitometry using the Scion Image software. The presence of linearity between the time of X-ray film exposure and the optical density of the glucose transporter bands was initially ensured (31).
Insulin Signaling Protein Analysis
Skeletal muscle preparation. Homogenates were prepared as previously described (21). Briefly, skeletal muscle was powdered under liquid nitrogen and suspended in three volumes of cell lysis buffer (Cell Signaling Technology, Beverly, MA). The samples were homogenized with a hand-held homogenizer for 1–2 min at one-half speed followed by 30 min of incubation on ice. The samples were then subjected to further homogenization with 20 up-and-down strokes using a tight-fitting Potter-Elvehjem homogenizer. These homogenates were then centrifuged at 10,000 rpm in 4°C for 10 min and stored at –70°C until Western blot analysis was undertaken.
Western blot analysis.
Membranes blocked in 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween-20 (PBST) for 1 h were incubated overnight with gentle agitation at 4°C with the specific primary antibody [at 1:1,000 dilution for phosphorylated (p)Akt, SHP2; 1:100 dilution for PKC and pPKC (Thr410), Cell Signaling Technology; 1:2,500 for protein-tyrosine-phosphatase 1B (PTP1B), BD Transduction Laboratories, Lexington, KY; and 1:4,000 dilution for vinculin, Sigma Chemical, St. Louis, MO]. The membranes were then washed with PBST three times for 15 min each. The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. After the membranes were washed three times for 15 min each, protein bands were visualized using the enhanced chemiluminescence method (Amersham Biosciences, Piscataway, NJ). The quantification of protein bands was performed by densitometry using the Scion Image software. The presence of linearity between the time of X-ray film exposure and the optical density of the various protein bands was initially ensured (21).
PKC Enzyme Activity Assay
PKC enzyme activity assay was performed as previously described (21). One milligram of skeletal muscle homogenate was precleared by incubation with 50 µl of 50% slurry of protein A/G-agarose (Santa Cruz Biotechnology) for 2 h at 4°C. Five micrograms of anti-PKC rabbit polyclonal antiserum raised against a peptide in the T-loop of PKC were incubated with 50 µl of 50% slurry of protein A/G-agarose for 2 h at 4°C on the rotor. The antibody-bound beads were washed three times with the cell lysis buffer and incubated with the precleared homogenate overnight at 4°C on the rotor. The precipitated agarose beads were then washed four times with the lysis buffer followed by two washes with the kinase buffer and resuspended in kinase buffer without myelin basic protein (MBP; substrate) and ATP. In vitro kinase assay was carried out for 30 min at 25°C in 50 µl of buffer containing 35 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM EGTA, 2 mM Na3VO4, 20 µg/ml leupeptin, 0.5 mM ATP, 4 µg MBP, and 0.4 µCi [
-32P]ATP. After incubation, 32P-labeled substrate was trapped on P81 filter papers. The P81 filter papers were then washed four times with 0.75% phosphoric acid and once with acetone and counted in a liquid scintillation counter. Samples that contained no antibody or no substrate were used as controls to account for background and endogenous phosphorylation, respectively.
Data Analysis
All data are expressed as means ± SE. Analysis of variance models were used to compare F2 treatment groups, and F values determined. Once significance was observed, intergroup differences were determined by the Fisher's paired least significant difference test, and significance was assigned when P values were <0.05.
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RESULTS |
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Birth weight in the pre- and postnatal nutrient-restricted group (F2 IUGR) was similar to the controls (F2 CON) (Table 1). Figure 2 depicts an age-dependent increase in body weight from birth to 15 mo of age. This increase in body weight is reflective of the postnatal growth pattern seen in F2 IUGR that was similar to F2 CON.
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Baseline fasting plasma glucose and insulin concentrations in the F2 IUGR group were significantly higher (P < 0.03 and P < 0.006, respectively; Table 2). The glucose/insulin ratio was significantly lower in the F2 IUGR group, supporting insulin resistance (P < 0.004; Table 2). There was no difference in baseline leptin or corticosteroid concentrations between the F2 IUGR and F2 CON (Table 2).
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During glucose tolerance testing the F2 IUGR group was mildly hyperglycemic. The plasma glucose concentrations in the F2 IUGR group were greater than those in the F2 CON at 5 min (P < 0.02) and at 15 min (P < 0.01), resulting in a higher glucose area under the curve (AUC) (P < 0.002) (Fig. 3, A and B). This hyperglycemia in the F2 IUGR group was associated with a significant increase in the insulin response, resulting in a 60% increase in the insulin AUC (P < 0.01; Fig. 4, A and B). Plasma insulin concentrations in the F2 IUGR group measured 15 min after the glucose challenge was significantly greater than the control (P < 0.004; Fig. 4A). The glucose-to-insulin ratio in F2 IUGR was significantly decreased at 5 and 15 min (P < 0.04 and P < 0.05, respectively) after glucose challenge compared with F2 CON (Fig. 5).
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The magnitude of decrease in plasma glucose after a single intravenous bolus of insulin at the dose employed in F2 IUGR was similar to the F2 CON group (Fig. 6A). The glucose AUC in the F2 IUGR was 186.4 ± 3.2 mg·dl–1·min–1, which was comparable with that of the F2 CON group (192 ± 4.6 mg·dl–1·min–1; Fig. 6B).
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The hepatic glucose production measured from the fraction of unlabeled glucose (M0 glucose) during intravenous glucose tolerance test (IVGTT) in the F2 IUGR group was significantly increased at 5 and 15 min after the glucose challenge (Fig. 7A). This resulted in a 30% increase in hepatic glucose production AUC in F2 IUGR (P < 0.01) compared with the F2 CON group (Fig. 7B).
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Examination of skeletal muscle total GLUT4 protein concentration revealed a significant increase in the 15-mo-old F2 IUGR group (P < 0.04) compared with F2 CON (Fig. 8A). The subcellular distribution of skeletal muscle GLUT4 in the basal state demonstrated lower PM-associated GLUT4 (P < 0.05) in F2 IUGR compared with F2 CON (Fig. 8B).
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Skeletal muscle pAkt concentrations were higher in the 15-mo-old F2 IUGR (P < 0.05) compared with F2 CON (Fig. 9A). In contrast, although no difference was observed in total PKC protein concentration (Fig. 9B), a decrease in pPKC concentration was observed in the F2 IUGR group (P < 0.05) vs. the F2 CON group (Fig. 9C). Paralleling the pPKC results, a decline in PKC enzyme activity (P < 0.05) was observed in the F2 IUGR vs. the CON group (Fig. 9D). The total SHP2 (Fig. 10A) and PTP1B (Fig. 10B) concentrations were unchanged in the F2 IUGR compared with the CON group.
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DISCUSSION |
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Various investigations have suggested that culturing preimplantation embryos in a glucose-rich medium can epigenetically alter the phenotype, as described in the "big baby syndrome" outcome of in vitro fertilization (12). Therefore, precautions were taken in this study to avoid this confounding variable by placing all embryos, those obtained from both nutrient-restricted and control mothers, in medium for less than 2 h, the minimum time required prior to the execution of embryo transfer. This precaution led to no intergroup differences in birth weight or growth pattern and circulating leptin or corticosterone concentrations, negating any major adverse effect of the limited culturing conditions employed in this investigation. In addition, culling a litter to contain six pups was targeted at standardizing postnatal nutrition. Therefore, any effect on the ultimate phenotype of the offspring is not reflective of culturing conditions prior to implantation, intrauterine, or postnatal nutritional factors. Changes, if observed in the F2 offspring, can therefore only reflect inheritance from F1 mothers that were exposed to pre- and postnatal nutrient restriction vs. mothers exposed to pre- and postnatal ad libitum nutrient intake.
Previous experiments defining transgenerational persistence of dysregulated glucose/insulin homeostasis consisted of F2 pups born naturally to the intrauterine selective nutrient (protein)-restricted (38) or uteroplacental-insufficient (4) F1 generation, where the F2 offspring mimicked mothers. The study with maternal nutrient restriction demonstrated no effect on the birth weight or glucose tolerance at 110 days of age with a decline in circulating insulin concentrations in the F2 IUGR (referred to as "RR") females, which is suggestive of an insulin-sensitive state, but an increase in the IUGR males consistent with insulin resistance (38). Islet cell transplantation in a diabetic pregnancy was previously observed to normalize circulating insulin concentrations in the adult F2 offspring (23). Uteroplacental insufficiency led to gestational diabetes in the F1 IUGR mothers with associated higher birth weights, hyperinsulinemia, and insulin resistance early during the suckling phase in the F2 offspring, followed later by a decline in pancreatic production of insulin and glucose intolerance (4). This study, although not sex-specific, supports the earlier emergence of insulin resistance rather than the reduction in F2 pancreatic -islets (8). These investigations collectively demonstrate that the intrauterine metabolically adverse environment contributes to the ultimate phenotype of the F2 offspring.
In the present investigation that focused primarily on the female, metabolic abnormalities persisted in the F2 offspring, despite normalizing the in utero metabolic environment of nutrient-restricted fetuses, by transferring embryos into normal females on the first day of conception. This supports factors beyond gestational diabetes seen in uteroplacental insufficiency-exposed F1 IUGR mothers (4) or the aberrant intrauterine metabolic environment of nutrient-restricted hypoinsulinemic F1 IUGR females (38). Despite the hypoinsulinemic response to a glucose challenge noted in F1 IUGR females secondary to intrauterine and postnatal nutrient restriction, insulin resistance of hepatic glucose output (8) and skeletal muscle GLUT4 translocation (32) occurred, with the latter present at birth. Molecular mechanisms responsible for the "metabolic imprint" of insulin resistance were inherited by the F2 offspring, independent of the intrauterine environment, serving as the trigger that possibly incites hyperinsulinemia. Hyperinsulinemia in turn was associated with hepatomegaly, reflective of hepatocyte hyperplasia (11), glycogen rich, or fatty liver (25), along with aberrant metabolic homeostasis as assessed by our measurement of glucose kinetics.
The F2 IUGR group demonstrated a decline in the glucose-to-insulin (endogenous) ratio, supporting the presence of insulin resistance that was not detected by the insulin tolerance test, perhaps related to the overriding exogenous insulin dose administered. The F2 IUGR group also demonstrated unchecked HGP despite hyperinsulinemia, with a trend toward increased glucose futile cycling. These perturbations in the face of no change in the rate of glucose clearance support hepatic insulin resistance.
Since skeletal muscle is the major tissue expressing insulin-responsive glucose uptake, this tissue was examined in our present investigation. In skeletal muscle under normal physiological circumstances, the rate-limiting step in glucose utilization is glucose transport. Hyperinsulinemia was associated with an increase in skeletal muscle GLUT4 expression reflected as total GLUT4 protein concentrations. This observation is in contrast to the decline in total GLUT4 protein concentration reported previously in the F1 IUGR (4) and the decrease in total GLUT4 mRNA in the naturally born F2 offspring to gestationally diabetic F1 IUGR females (4). Despite the increased total GLUT4 expression, a significant decline in basal PM-associated GLUT4 pool was noted in the F2 IUGR group. This decline in PM to total GLUT4 concentration ratio in the F2 IUGR supports skeletal muscle insulin resistance. Although basal PM-associated GLUT4 concentrations increased, this finding in principle parallels the insulin resistance of skeletal muscle GLUT4 translocation that we previously observed in the F1 IUGR female skeletal muscle (32).
Our quest for mechanisms in the postinsulin receptor signaling pathway responsible for this dichotomy consisting of increased expression of total GLUT4 and decreased plasma membrane GLUT4 concentration yielded an increase in pAkt and a decrease in pPKC and enzyme activity, respectively. The increase in insulin signaling seen as higher pAkt concentration may underlie the ultimate increased total GLUT4 expression, whereas the associated downregulation of plasma membrane GLUT4 concentration due to either defective translocation or internalization may relate to reduced activated PKC. A similar dichotomy between pAkt and pPKC is also observed in the F1 IUGR skeletal muscle, as previously reported (21).
Thus features of insulin resistance are far more exaggerated in the F2 offspring compared with the F1 generation (8, 21, 32), since the latter demonstrated no change in HGP despite hypoinsulinemia supporting emerging hepatic insulin resistance. In addition, skeletal muscle GLUT4 changes in F2 are further exaggerated compared with F1 IUGR. Therefore, our present observations, along with our previous F1 investigation, support the transgenerational persistence of hepatic and skeletal muscle insulin resistance that is further amplified to what was observed in the F1 generation (8, 21, 32). Similarly, previously reported persistent hyperglycemia in Goto kakizaki embryo reared in euglycemic (Wistar) uterine environment (9) strengthens the findings from our present investigation demonstrating an inability to alter strong genetic factors in the development of type 2 diabetes. Our interpretation of the present observations in the IUGR F2 female offspring is that, despite providing a normal intrauterine and postnatal nutritional/metabolic environment, the offspring expressed mild glucose intolerance, hyperinsulinemia, and hepatic and skeletal muscle insulin resistance. Since no genetic mutations were present in control Sprague-Dawley rats that were used as surrogate mothers in the study, epigenetic perturbations instilled in the F1 generation due to fetal and postnatal exposure to aberrant nutritional state may be inherited by the F2 offspring. This inheritance pattern was not erased by a normal intrauterine and postnatal nutritional environment.
Although the role of epigenetic regulation of gene expression has been extensively investigated in cancer biology (29) and to a limited extent in metabolic programming, most of the studies to date have been associative in nature (16, 18, 22, 30). Global DNA methylation (22) or gene promoter-specific DNA methylation changes (16) have been observed in response to nutritional aberrations. Some of these changes have been reported to be tissue specific in the F1 generation, spanning only the suckling phase (18). Some of the DNA methylation changes have been associated with alterations of the chromatin histone code along with perturbed gene expression patterns (30). However, these investigations fail to demonstrate a cause and effect relationship and do not place these tissue-specific changes in the context of whole body physiology (18), nor do they explore the F2 generation, lending controversy to the theory of DNA methylation in metabolic programming. This controversy is exemplified given various other heritable mechanisms consisting of corepressor complexes involved in altering the histone code, microRNAs, and other gene modifiers that have been described (5, 10). Our investigations are the first to examine the glucose/insulin homeostasis in the context of whole body physiology along with skeletal muscle insulin signaling and GLUT4 distribution in the F2 generation reared in a normal intrauterine and postnatal nutritional environment. Although speculative, our investigation suggests a role for epigenetic mechanisms in the persistence of metabolic aberrations in the F2 generation of the IUGR group. The specific constitution of these contributing heritable mechanisms requires future investigation.
The overlay of gestational diabetes that may develop in the IUGR adult female (4) on these inherent mechanisms may further contribute toward the transgenerational persistence of an abnormal metabolic phenotype and amplify the burden of type 2 diabetes mellitus. Acute or chronic malnutrition of mothers that rampantly occurs in developing countries can impair normal fetal development and induce impairment of glucose regulatory mechanisms in the offspring. This combination of a latent diabetogenic tendency and the metabolic stress of pregnancy may result in gestational diabetes that in turn may induce impaired glucose tolerance and gestational diabetes in the next generation. A chain reaction of transmission of gestational diabetes from one generation to another is thereby initiated. By transferring embryos with a latent diabetogenic tendency to a normal intrauterine environment and avoiding gestational hyperglycemia we have shown features consistent with persistence of insulin resistance in the F2 offspring.
Previous investigations using various therapeutic agents, such as insulin-like growth factors (36) and growth hormone (37) during adult life and glucagon-like peptide-1 agonists (26) and leptin (35) during the early developmental stages of the F1 generation, were targeted at amelioration of the adult phenotype. However, based on our current findings, this phenotype correction may have a temporary effect limited to only the generation receiving the intervention. Nutrition-induced heritable changes involving dysregulation of glucose/insulin homeostasis and consequences on skeletal muscle GLUT4 are likely transmitted to the F2 generation despite correction of nutritional deficiencies in the F1 generation. Similarly controlling gestational diabetes alone may prove to be inadequate in preventing transgenerational heritable transmission of the diabetogenic propensity.
Extrapolating our current findings to the human epidemic of type 2 diabetes in the developed and developing world raises major concerns. Malnutrition during pregnancy or lactation is experienced by many individuals worldwide. Nutritional deprivation during this critical period of development may cause malprogamming and adversely alter not only the F1 generation but the phenotype of future generations. Implementation of corrective nutritional programs targeted at the F1 generation alone may be therapeutic but not entirely successful in ameliorating inheritance to subsequent generations. Overlaying this inheritance probability with lifestyle factors that include high-calorie diets and a sedentary lifestyle may further perpetuate the propensity toward type 2 diabetes. This may contribute toward the worldwide epidemic of type 2 diabetes mellitus and the earlier presentation of insulin resistance and type 2 diabetes mellitus. Reversal of this trend may require future investigation of specific heritable mechanisms and therapeutic interventions targeted at these mechanisms.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* These authors contributed equally to this work. 
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P < 0.01, IUGR (n = 5) compared with CON (n = 6). Values are means ± SE.
