Maternal Low Protein Research Articles

Adipocyte pyruvate dehydrogenase kinase 4 expression is associated with augmented PPARγ upregulation in early-life programming of later obesity

We studied adipocytes from 8-week-old control rat offspring (CON) or rat offspring subjected to maternal low (8%) protein (MLP) feeding during pregnancy/lactation, a procedure predisposing to obesity. Acute exposure to isoproterenol or adenosine enhanced PDK4 and PPARγ mRNA gene expression in CON and MLP adipocytes. Enhanced adipocyte Pdk4 expression correlated with increased PPARγ expression. Higher levels of PDK4 and PPARγ were observed in MLP adipocytes. SCD1 is a PPARγ target. Isoproterenol enhanced adipocyte PDK4 and SCD1 gene expression in parallel. This could reflect augmented PPARγ expression together with enhanced lipolytic stimulation to supply endogenous PPARγ ligands, allowing enhanced adipocyte PDK4 and SCD1 expression via PPARγ activation. In contrast, the effect of adenosine to increase PDK4 expression is independent of stimulation of lipolysis and, as SCD1 expression was unaffected by adenosine, unlikely to reflect PPARγ activation. Increased adipocyte expression of both PDK4 and SCD1 in the MLP model could participate as components of a “thrifty” phenotype, favouring the development of obesity.

Maternal Periconceptional and Gestational Low Protein Diet Affects Mouse Offspring Growth, Cardiovascular and Adipose Phenotype at 1 Year of Age

Human and animal studies have revealed a strong association between periconceptional environmental factors, such as poor maternal diet, and an increased propensity for cardiovascular and metabolic disease in adult offspring. Previously, we reported cardiovascular and physiological effects of maternal low protein diet (LPD) fed during discrete periods of periconceptional development on 6-month-old mouse offspring. Here, we extend the analysis in 1 year aging offspring, evaluating mechanisms regulating growth and adiposity. Isocaloric LPD (9% casein) or normal protein diet (18% casein; NPD) was fed to female MF-1 mice either exclusively during oocyte maturation (for 3.5 days prior to mating; Egg-LPD, Egg-NPD, respectively), throughout gestation (LPD, NPD) or exclusively during preimplantation development (for 3.5 days post mating; Emb-LPD). LPD and Emb-LPD female offspring were significantly lighter and heavier than NPD females respectively for up to 52 weeks. Egg-LPD, LPD and Emb-LPD offspring displayed significantly elevated systolic blood pressure at 52 weeks compared to respective controls (Egg-NPD, NPD). LPD females had significantly reduced inguinal and retroperitoneal fat pad: body weight ratios compared to NPD females. Expression of the insulin receptor (Insr) and insulin-like growth factor I receptor (Igf1r) in retroperitoneal fat was significantly elevated in Emb-LPD females (P<0.05), whilst Emb-LPD males displayed significantly decreased expression of the mitochondrial uncoupling protein 1 (Ucp1) gene compared to NPD offspring. LPD females displayed significantly increased expression of Ucp1 in interscapular brown adipose tissue when compared to NPD offspring. Our results demonstrate that aging offspring body weight, cardiovascular and adiposity homeostasis can be programmed by maternal periconceptional nutrition. These adverse outcomes further exemplify the criticality of dietary behaviour around the time of conception on long-term offspring health.

Chronic Maternal Protein Deprivation in Mice Is Associated with Overexpression of the Cohesin-Mediator Complex in Liver of Their Offspring

Epigenetic mechanisms may play an important role in the developmental programming of adult-onset chronic metabolic diseases resulting from suboptimal fetal nutrition, but the exact molecular mechanisms are incompletely understood. Given the central role of the liver in metabolic regulation, we investigated whether chronic maternal dietary protein restriction has long-term effects on liver gene expression in the offspring. We fed adult C57BL/6J dams ad libitum an 8% maternal low-protein (MLP) or 20% protein control diet (C) from 4 wk prior to mating until the end of lactation. Male pups were weaned to standard nonpurified diet and singly housed at 21 d of age (d 21). Body weights were followed to 1 y of age (1 y). At d 21 and 1 y, organs were quantitatively dissected and analyzed. MLP offspring had significantly lower body weights at all ages and significantly lower serum activity of alanine aminotransferase and lactate dehydrogenase at 1 y. Gene expression profiling of liver at 1 y showed 521 overexpressed and 236 underexpressed genes in MLP compared to C offspring. The most important novel finding was the overexpression of genes found in liver that participate in organization and maintenance of higher order chromatin architecture and regulation of transcriptional activation. These included members of the cohesin-mediator complex, which regulate gene expression by forming DNA loops between promoters and enhancers in a cell type-specific fashion. Thus, our findings of increased expression of these factors in liver of MLP offspring implicate a possible novel epigenetic mechanism in developmental programming.

Protein restriction during gestation alters histone modifications at the glucose transporter 4 (GLUT4) promoter region and induces GLUT4 expression in skeletal muscle of female rat offspring

Maternal nutrition during pregnancy is an intrauterine factor that results in alteration of the offspring genome and associates with disease risk in the offspring. We investigated the impact of a maternal low-protein (LP) diet on the expression of glucose transporter 4 (GLUT4) in offspring skeletal muscle. GLUT4 is an insulin-regulated glucose transporter involved in insulin sensitivity and carbohydrate metabolism in muscle cells. We observed sex-dependent GLUT4 mRNA expression and increased GLUT4 protein content in female pup skeletal muscle with maternal LP. Analysis of transcriptional and epigenetic regulation of increased skeletal muscle GLUT4 expression in offspring rats revealed the regulatory mechanisms involved. The protein level of myocyte enhancer factor 2A (MEF2A), which has been known as an activator of GLUT4 transcription via the ability to carry out specific binding to the GLUT4 MEF2 binding sequence, increased in female pups whose mothers were fed a LP diet. Modifications of chromatin structure, including acetylated histone H3, acetylated histone H4 and di-methylated histone H3 at lysine 4, were detected at a significantly increased level at the GLUT4 promoter region in female pup muscle following a maternal LP diet. Glycogen content was also detected as up-regulated, accompanied by increased glycogen synthase in LP female offspring muscle. These results document that maternal protein restriction during pregnancy induces GLUT4 expression in female offspring skeletal muscle but not in males, which may indicate sex-dependent adaptation of glucose metabolism to a maternal LP diet.

Maternal protein restriction in the rat during pregnancy and/or lactation alters cognitive and anxiety behaviors of female offspring

Maternal protein deficiencies can developmentally program offspring to lifelong dysfunction of many physiological systems. We hypothesized that maternal isocaloric low protein diet during fetal and early postnatal development would negatively affect female offspring anxiety, exploration, associative learning and motivation as measured by the elevated plus maze (EPM), open field test (OFT), operant conditioning and the progressive ratio task, respectively. Control mothers (C) received a 20% casein diet and restricted mothers (R) a 10% casein diet to provide four groups: CC, RR, CR, and RC (first letter pregnancy diet and second lactation diet) to enable evaluation of offspring effects influenced by maternal diet during pregnancy and lactation. Maternal protein restriction decreased open arm time and distance in RR and RC offspring, increased anxiety behavior, in the EPM. In the OFT, the RR and RC offspring displayed decreased exploration (increased stress) as indexed by decreased distance in the center zone. These behaviors in the EPM and OFT was associated with increased corticosterone levels during an immobilization test in the RR offspring with intermediary effects in the RC offspring. Learning impairment was observed in the RR, CR and RC offspring during fixed ratio 5 schedule of reinforcement. Motivational effects were measured in RR offspring responding less, decreased motivation, and CR offspring making more responses, increased motivation, than CC offspring. These findings reveal the negative effects of developmental protein restriction on female offspring behavior. The underlying basis for these negative outcomes remains to be elucidated.

Emergence of ageing-related changes in insulin secretion by pancreatic islets of male rat offspring of mothers fed a low-protein diet

Maternal low-protein (LP) diets programme β-cell secretion, potentially altering the emergence of ageing of offspring pancreatic function. We hypothesised that isolated pancreatic islet β-cell secretory responses are blunted in offspring exposed to LP during development and age-related reduction is influenced by the developmental stage of exposure to decreased nutrition. We studied male offspring of rats fed control (C) or LP protein (R) diets in pregnancy, first letter and/or lactation second letter of CC, RR, CR or RC groups. Serum glucose, insulin and homeostatic model assessment (HOMA) were measured. Pancreatic islets were isolated and in vitro insulin secretion quantified in low (LG - 5 mM) or high glucose (HG - 11 mM). Body weight and serum values between groups were similar at all ages. Insulin and HOMA rose with age and were highest at postnatal day (PND) 450 in all groups. At PND 36, insulin secretion was greatest in RR and RC. Only CC increased insulin secretion to HG. By PND 110, restricted groups responded less to LG but increased secretion to HG. By PND 450, CC offspring alone increased secretion to HG. Despite minimal differences in circulating insulin and glucose, reduced maternal protein intake affected insulin secretion at all ages. In addition, ageing reduced function in all R groups compared with CC by PND 110 and further by PND 450 most markedly in RC. We conclude that maternal LP diet during pregnancy and/or lactation impairs offspring insulin secretory response to a glucose challenge and alters the trajectory of ageing of pancreatic insulin secretion.

Effects of a maternal diet supplemented with chocolate and fructose beverage during gestation and lactation on rat dams and their offspring

1. Consumption of a high-fat and high-energy diet during pregnancy leads to a risk of long-term consequences on fetal development, as well as on the postnatal health of offspring. To investigate the effects of such a diet on fetal programming, we established a high-energy intake pregnant rat model using chocolate and fructose beverage as supplements to a normal chow diet. 2. Pregnant Sprague-Dawley rats were assigned to either chow (control) or a diet supplemented with chocolate and fructose beverage throughout gestation and lactation. The male F(1) pups received normal chow diet after weaning. Physiological or pathological changes in dams and pups (e.g. glucose and lipid metabolism) were evaluated. 3. The results showed that dams offered the high-fat (mainly from chocolate) and high-calorie diet during gestation consumed more energy and gained more weight than chow-fed dams. Over-consumption of chocolate reduced chow intake in dams, leading to low maternal protein supply. As a result, pups from these dams exhibited reduced birth weight that lasted until adulthood. The high-energy diet during lactation led to increased total body fat, as well as impaired liver function, in offspring; thus, the lactational diet is suggested to be a stronger determinant of offspring fat metabolism than gestational diet. 4. The results of the study suggest that over-supply of carbohydrates, such as chocolate and fructose, either during gestation or lactation has a negative impact on the well-being of offspring.

Adverse cardiac remodeling due to maternal low protein diet is associated with alterations in expression of genes regulating glucose metabolism

Background and aimsWe have previously shown that a maternal low protein (LP) diet during pregnancy in the rat results in adverse ventricular remodeling and contractile deficiencies of the neonatal rat heart. Since pathological cardiac hypertrophy is associated with increased expression of genes involved in glucose handling, this study was undertaken to examine if maternal LP diet alters the expression of genes encoding for some key components of glucose metabolism and uptake, and of the insulin receptor (IR) signal transduction in the heart of male offspring. Methods and resultsWe determined the effect of maternal LP and normal diet (90 and 180 g/casein/kg respectively) on IR β-subunit, insulin receptor substrate (IRS)-1, phosphotyrosyl protein phosphatase (PTP) 1B, GLUT4 and phosphatidylinositol (PI) 3-kinase in male rat offspring at 24 h and at 1, 4 and 8 wks post-partum. Quantitative real-time RT-PCR revealed significant age-dependent increases in the expression of IR β-subunit, IRS-1, PTP1B, GLUT4 and PI3-kinase in the LP group with concomitant increases in corresponding protein abundance at 4 wks of age. These changes were associated with increases in left ventricular (LV) internal diameters as well as increases in LV wall thickness. ConclusionA maternal LP diet can induce increases in the gene expression and protein levels of key components of glucose metabolism and the IR signal transduction pathway in the neonatal rat heart, which may be related to accelerated energy supply, demand and utilization for ventricular remodeling due to compromised contractile performance during early life.

Gestational low protein diet selectively induces the amino acid response pathway target genes in the liver of offspring rats through transcription factor binding and histone modifications

The amino acid response (AAR) pathway detects a deficiency of dietary amino acid or protein. To investigate the impact of gestational protein restriction on the AAR pathway in offspring, pregnant Sprague–Dawley rats were fed a control (C) or low protein (LP) diet during gestation. Livers of female offspring were collected on postnatal d 38. The mRNA amount of Atf3 in LP offspring increased significantly compared with C offspring, while Asns did not differ between the two groups. ATF4 and p-eIF2α were both induced in LP offspring, whereas p-ERK was significantly decreased. Additionally, amino acid limitation (− AA) in HepG2 cells increased p-ERK and AAR pathway-related genes, while U0126 decreased p-ERK but did not completely reverse the activation of AAR pathway-related genes. Chromatin immunoprecipitation assay demonstrated an increased association of both RNA polymerase II (Pol II) and ATF4 at the Atf3 promoter in LP offspring, while acetylated histone H4, tri-methyl histone H3 at lysine 9, ATF4, ATF3, C/EBPβ, and CHOP, but not Pol II, were all increased at the Asns promoter in LP offspring. In − AA HepG2 cells, C/EBPβ siRNA treatment did not prevent the activation of either ATF3 or ASNS in response to −AA, while ATF4 siRNA prevented the activation of ASNS but not ATF3. Our data demonstrates that a maternal LP diet programs the AAR pathway in the liver of offspring rats. The differential priming of the downstream target genes of the AAR pathway in response to maternal LP diet presents a novel regulatory mechanism related to nutrient–gene interactions.

Metabolic adaptations to early life protein restriction differ by offspring sex and post-weaning diet in the mouse

Background and aimsLow birth weight affects 1 in every 7 babies born globally and can predict a lifetime of increased risk for adverse health outcomes, including cardiovascular disease, hypertension, obesity, diabetes, and metabolic syndrome. Maternal low protein diet during pregnancy and lactation is a well-characterized rat model for low birth weight and the subsequent increase in chronic disease risk. However, mice have been relatively understudied in this paradigm and represent a critical resource for investigating the underlying molecular mechanisms that link adverse early life experience and the development of chronic disease. Methods and resultsThe present manuscript describes a mouse model of low birth weight (maternal consumption of low protein diet (8% protein) through pregnancy and lactation) and characterizes metabolic adaptations (food intake, locomotor activity, oxygen consumption, and glucose tolerance) in male and female offspring. At weaning, mice were maintained either on the control diet or a high fat diet. Notable sex differences were observed, with male mice from the low protein pregnancies showing increased food intake, hyperactivity and increased metabolic rate only when weaned to the high fat diet, while female mice consistently showed increased food intake and were hypometabolic, regardless of post-weaning diet. ConclusionThese data identify offspring sex and post-weaning diet as critical variables in the metabolic adaptations to early life protein deficiency, and suggest that females may be more vulnerable to the adverse long-term health consequences of low birth weight

Pre- and/or postnatal protein restriction developmentally programs affect and risk assessment behaviors in adult male rats

Developmental programming resulting from a suboptimal intrauterine environment can predispose offspring to a wide-range of lifelong health complications. Little is known about the effects maternal protein restriction during pregnancy and/or lactation has on offspring neurodevelopment. We hypothesized that maternal isocaloric low protein diet during pregnancy and/or lactation would negatively influence male offspring affect and risk assessment behaviors as measured by elevated plus maze and open field tests. Control mothers received 20% casein (C) and restricted mothers (R) 10% casein to provide four groups: CC, RR, CR, and RC (first letter pregnancy diet and second letter lactation diet) to evaluate effects of maternal diet on offspring risk assessment, anxiety and exploratory behaviors. Elevated plus maze results showed an effect of pre- and/or postnatal diet manipulation in open arm time ( p < 0.05) with increases seen in the RR (157 ± 22.7 s), CR (137 ± 23.2 s) and RC (146.8 ± 10.8 s) offspring relative to CC (52 ± 8.6 s) offspring. This behavior indicates decreased avoidance (less anxiety) and increased exploration by experimental groups. However, in the open field test the RR (17 ± 4.2 entries) offspring entered the center zone less than the CC (35 ± 6.6 entries) offspring thus exhibiting increased anxiety with no other groups showing effects. Elevated levels of corticosterone were measured before, during and after immobilization in the RR compared to CC offspring. These findings show protein restriction during critical periods of development negatively program offspring behavior. The underlying anatomical structures affected remain to be elucidated.

A maternal low protein diet programs glucose and fatty acid metabolism differentially in adult male and female mouse offspring

Nutritional conditions during fetal life can influence the risk to develop the metabolic syndrome in adult life (‘metabolic programming’). We aimed to establish a mouse model of metabolic programming focusing on the effects of a maternal low protein diet during gestation on glucose and lipid metabolism in the adult offspring. Pregnant C57BL/6J mice received a control or a low protein diet throughout gestation. Offspring received a low fat diet or a high fat diet from 6–22 weeks of age. Glucose metabolism was studied with a whole‐body‐glucose test using [6,6‐2H]‐glucose. Hepatic gene expression was characterized by microarray. Maternal low‐protein‐diet did not affect glucose metabolism in male offspring. Female offspring from normal‐protein fed dams was relatively resistant to diet‐induced metabolic dysregulation. Maternal low‐protein‐diet during gestation led to deteriorated insulin sensitivity upon high‐fat feeding in female offspring. Microarray analyses showed a sex‐specific effect of the maternal diet. We conclude that, in mice, maternal protein restriction during gestation does not change the glucose response to a high fat diet in male offspring. However, it changes fatty acid and glucose metabolism in female offspring in such a way that it resembles male metabolism. Supported by the Netherlands Heart Foundation, grant 2004T048.

Maternal protein restriction during pregnancy induces CCAAT/enhancer-binding protein (C/EBPβ) expression through the regulation of histone modification at its promoter region in female offspring rat skeletal muscle

Maternal nutrition during pregnancy is an important intrauterine environmental factor that can cause persistent alterations of the offspring genome and is associated with potential disease risk later in life. In the present study, we investigated the impact of a maternal low protein diet (LP) on the expression of the transcription factor CCAAT/enhancer-binding protein (C/EBPβ) in offspring skeletal muscle. C/EBPβ belongs to a family of transcription factors that regulates the expression of genes involved in energy homeostasis and muscle development. We investigated C/EBPβ transcriptional regulation from an epigenetic aspect. We observed sex-dependent differences in C/EBPβ expression in offspring skeletal muscle subjected to a maternal protein-restricted diet. In female offspring skeletal muscle, both C/EBPβ mRNA and protein levels were increased by maternal protein restriction. However, C/EBPβ expression was not altered in other tissues or male offspring. Analysis of transcriptional and epigenetic regulation showed acetylated histone 3 and acetylated histone 4 at significantly increased levels at the C/EBPβ promoter region in female LP pup's muscle. Phosphoenolpyruvate carboxykinase (PEPCK) gene transcription was also up-regulated in female LP pups through the increased binding of C/EBPβ at its promoter. The induction of C/EBPβ expression in female offspring skeletal muscle by maternal protein restriction during pregnancy may indicate C/EBPβ involvement in signaling response in energy metabolism to a low maternal protein diet.

The efficacy of estrogen replacement therapy for vascular dysfunction induced by a maternal low protein diet

背景: 妊娠中の母体の低蛋白栄養により, 児の成人期での高血圧, 冠血管障害, 糖尿病などの成人病が発症しやすくなることが知られている. さらに動物実験では雌より雄に高血圧が発症しやすくなることが明らかになっている. 過去にわれわれは卵巣を摘出し, 低エストロゲン状態にした母体低蛋白栄養のラットを用いた実験で, コントロール群に比べ, 母体低蛋白栄養群では高血圧や冠動脈周囲の線維化が有意に進行し, 卵巣摘出群でさらに顕著であることを証明している. また, wire myographを用いた血管機能評価では, ブラジキニンに対する血管拡張反応が有意に障害されたことから, 母体低蛋白栄養による高血圧や冠動脈血管周囲の線維化にはブラジキニンが関与していることも明らかにしてきた. そこで, 今回の実験は妊娠中の母体低蛋白栄養によるfetal programmingにより誘導された血管内皮細胞機 能障害がエストロゲン補充により改善することを研究仮説として実験を行った. 方法: Wistar ratを交配し, 妊娠中2つの栄養群;コントロール群 (C), 低蛋白栄養群 (R) に分け飼育した. 分娩後, 両群とも通常餌とし, 仔は日齢50に各群に対し次の3種の手術を行い, 合計6群を作成した;sham手術群 (CO, RO), 卵巣摘出群 (CX, RX), エストロゲン補充群 (CE, RE). 日齢50,100,150,175に血圧を測定し, 日齢175に腸間膜動脈を摘出しwire myographを用いて血管機能評価を行った. 結果: 血圧の変化はいずれの日齢においても各群間に有意差を認めなかった. wire myographにおいては, ブラジキニンに対する血管拡張反応がCOに対しRO, RXで有意に鈍化し, REで有意に改善を認めた. 結論: 母体低蛋白栄養で起こる血管機能障害における性差発現にはエストロゲンが関与していることが再確認された. また, 引き起こされた血管機能障害はエストロゲンを補充することで代償することができる可能性があることが示唆された.

Alteration of mitochondrial function in adult rat offspring of malnourished dams

Under-nutrition as well as over-nutrition during pregnancy has been associated with the development of adult diseases such as diabetes and obesity. Both epigenetic modifications and programming of the mitochondrial function have been recently proposed to explain how altered intrauterine metabolic environment may produce such a phenotype. This review aims to report data reported in several animal models of fetal malnutrition due to maternal low protein or low calorie diet, high fat diet as well as reduction in placental blood flow. We focus our overview on the β cell. We highlight that, notwithstanding early nutritional events, mitochondrial dysfunctions resulting from different alteration by diet or gender are programmed. This may explain the higher propensity to develop obesity and diabetes in later life.

Pre- and/or postnatal protein restriction in rats impairs learning and motivation in male offspring

Suboptimal developmental environments program offspring to lifelong health complications including affective and cognitive disorders. Little is known about the effects of suboptimal intra-uterine environments on associative learning and motivational behavior. We hypothesized that maternal isocaloric low protein diet during pregnancy and lactation would impair offspring associative learning and motivation as measured by operant conditioning and the progressive ratio task, respectively. Control mothers were fed 20% casein (C) and restricted mothers (R) 10% casein to provide four groups: CC, RR, CR, and RC (first letter pregnancy diet and second letter lactation diet), to evaluate effects of maternal diet on male offspring behavior. Impaired learning was observed during fixed ratio−1 operant conditioning in RC offspring that required more sessions to learn vs. the CC offspring (9.4±0.8 and 3.8±0.3 sessions, respectively, p<0.05). Performance in fixed ratio−5 conditioning showed the RR (5.4±1.1), CR (4.0±0.8), and RC (5.0±0.8) offspring required more sessions to reach performance criterion than CC offspring (2.5±0.5, p<0.05). Furthermore, motivational effects during the progressive ratio test revealed less responding in the RR (48.1±17), CR (74.7±8.4), and RC (65.9±11.2) for positive reinforcement vs. the CC offspring (131.5±7.5, p<0.05). These findings demonstrate negative developmental programming effects due to perinatal isocaloric low protein diet on learning and motivation behavior with the nutritional challenge in the prenatal period showing more vulnerability in offspring behavior.

Effect of a low-protein diet during pregnancy on expression of genes involved in cardiac hypertrophy in fetal and adult mouse offspring.

Gene markers for cardiomyocyte growth, proliferation and remodeling were examined in mouse fetuses and adult male offspring exposed to maternal low-protein (LP) diet during pregnancy. Whole heart volume, measured by magnetic resonance imaging, was smaller in day 15 LP fetuses v. those from chow-fed dams (C), whereas heart volume was greater in adult LP v. C offspring. These LP offspring were hypertensive and had larger cardiomyocytes v. C animals. The mRNA levels of cyclin G1, a marker for cell growth, were lower in LP fetal hearts v. C hearts, but similar in the left ventricle of adult LP and C offspring. Opposite trends were found in brain natriuretic peptide levels (a marker of cardiac hypertrophy). Thus, maternal LP during pregnancy results in smaller fetal hearts and is accompanied by changes in expression of genes involved in cardiomyocyte growth, which are associated with cardiac hypertrophy and hypertension in adulthood.

325 Iugr Affects Long Term Postischemic Neovascularization Capacity

Introduction: Cardiovascular disease is a major cause of premature death. The severity of such diseases has been suggested to result from insufficient angiogenesis processes. Low birth weight is now recognized as a risk factor of mortality for such disease. However, the vascular repair capacity after ischemia in IUGR adult offspring is unknown. Objective: To show how postischemic neovascularization capacity is affected in 12 mo-old adult IUGR rat offspring (maternal gestational low protein diet). Material and Methods: The unilateral hind limb ischemia model by femoral artery resection was used to study the neovascular capacity; the controlateral hind limb served as a reference for each animal. Laser Doppler imaging was used to assess blood flow perfusion in both hind limb after ischemia. Capillary densities of the anterior tibial muscle from both controlateral and ischemic limbs was measured at day 21. Results: After ischemia, IUGR offspring showed marked decreased blood flow in ischemic hind limb from day 1 (-60 % vs -40 % in IUGR and control offspring, p < 0.05). At day 21, while ischemia promoted neovascularization in control offspring (+ 22 %), vascular density was decreased in IUGR ischemic hind limb by 30 % (p < 0.05). Moreover, muscle capillary density of non-ischemic hind limb was higher in IUGR as compared with control offspring (+ 20 %, p < 0.05). Conclusion: Postischemic neovascularization capacity following hind limb ischemia is affected in IUGR adult rat offspring. Angiogenic repair pathways, more than basal capillary density, may be affected in such condition.

Gestational low protein diet in the rat mediates Igf2 gene expression in male offspring via altered hepatic DNA methylation

Biological responses to environmental stress, including nutrient limitation are mediated in part by epigenetic modifications including DNA methylation. Insulin-like growth factor II (Igf2) and H19 are subject to epigenetic modifications leading to genomic imprinting. The present study was designed to test the effect of maternal low protein diet on the Igf2/H19 locus in offspring. Pregnant Sprague-Dawley rats were fed diets containing 180 g/kg casein (control) or 90 g/kg (LP) casein with either 1 mg/kg (LP) or 3 mg/kg folic acid (LPF). LP diet increased Igf2 and H19 gene expression in the liver of day 0 male offspring and the addition of folic acid reduced the mRNA level in LPF rats to that of the control group. DNA methylation in Imprinting Control Region (ICR) of Igf2/H19 locus increased significantly following maternal LP diet but rats fed the LPF diet did not exhibit the hypermethylation. The Differential Methylation Region 2 (DMR2) did not show any change in methylation in either LP or LPF rats. The expression of Dnmt1 and Dnmt3a, the members of DNA methyltransferase family, and methyl CpG-binding domain 2 (Mbd2) was significantly increased following the maternal LP diet but did not differ between the control and LPF group. There is a strong correlation between methylation of ICR with the expression of Igf2 and H19. These results suggested that maternal exposure to a low protein diet and folic acid during gestation alters gene expression of Igf2 and H19 in the liver by regulating the DNA methylation of these genes. The DNA methyltransferase machinery may be involved into the programming of imprinted genes through the imprinted control region.

The Effects of Intrauterine Malnutrition on Maternal-Fetal Cholesterol Transport and Fetal Lipid Synthesis in Mice

Intrauterine malnutrition is associated with increased susceptibility to chronic diseases in adulthood. Growth-restricted infants display a less favorable lipid profile already shortly postnatal. Maternal low protein diet (LPD) during gestation is a well-defined model of fetal programming in rodents and affects lipid metabolism of the offspring. Effects of LPD throughout gestation on physiologic relevant parameters of lipid metabolism are unclear. We aimed to determine effects of LPD on maternal-fetal cholesterol fluxes and fetal lipid synthesis in mice. Pregnant mice (dams) were fed with a control (18% casein) or an LPD (9% casein) from E0.5 onward. We quantified maternal-fetal cholesterol transport and maternal cholesterol absorption at E19.5 using stable isotopes. We determined fetal lipid biosynthesis at E19.5, after administration of (1-C)-acetate from E17.5 onward. LPD did not change fetal and maternal plasma and hepatic concentrations of cholesterol and triglycerides. LPD affected neither the magnitudes of maternal-fetal cholesterol flux, maternal cholesterol absorption, nor fetal synthesis of cholesterol and palmitate (both groups, approximately 14% and approximately 13%, respectively). We conclude that LPD throughout gestation in mice does not affect maternal-fetal cholesterol transport, fetal cholesterol or fatty acid synthesis, indicating that programming effects of LPD are not mediated by short-term changes in maternal-fetal lipid metabolism.