Intrauterine growth restriction combined with a maternal high-fat diet increased adiposity and serum corticosterone levels in adult rat offspring.

Intrauterine growth restriction (IUGR) and fetal exposure to a maternal high-fat diet (HFD) independently increase the risk of developing obesity in adulthood. Excess glucocorticoids increase obesity. We hypothesized that surgically induced IUGR combined with an HFD would increase adiposity and glucocorticoids more than in non-IUGR offspring combined with the same HFD, findings that would persist despite weaning to a regular diet. Non-IUGR (N) and IUGR (I) rat offspring from dams fed either regular rat chow (R) or an HFD (H) were weaned to either a regular rat chow or an HFD. For non-IUGR and IUGR rats, this study design resulted in three diet groups: offspring from dams fed a regular diet and weaned to a regular diet (NRR and IRR), offspring rats from dams fed an HFD and weaned to a regular diet (NHR and IHR) and offspring from dams fed an HFD and weaned to an HFD (NHH and IHH). Magnetic resonance imaging or fasting visceral and subcutaneous adipose tissue collection occurred at postnatal day 60. IHH male rats had greater adiposity than NHH males, findings that were only partly normalized by weaning to a regular chow. IHH male rats had a 10-fold increase in serum corticosterone levels. IHH female rats had increased adiposity and serum triglycerides. We conclude that IUGR combined with an HFD throughout life increased adiposity, glucocorticoids and triglycerides in a sex-specific manner. Our data suggest that one mechanism through which the perinatal environment programs increased adiposity in IHH male rats may be via increased systemic glucocorticoids.

Altered expression and chromatin structure of the hippocampal IGF1r gene is associated with impaired hippocampal function in the adult IUGR male rat.

Exposure to intrauterine growth restriction (IUGR) is an important risk factor for impaired learning and memory, particularly in males. Although the basis of IUGR-associated learning and memory dysfunction is unknown, potential molecular participants may be insulin-like growth factor 1 (Igf1) and its receptor, IGF1r. We hypothesized that transcript levels and protein abundance of Igf1 and IGF1r in the hippocampus, a brain region critical for learning and memory, would be lower in IUGR male rats than in age-matched male controls at birth (postnatal day 0, P0), at weaning (P21) and adulthood (P120). We also hypothesized that changes in messenger Ribonucleic acid (mRNA) transcript levels and protein abundance would be associated with specific histone marks in IUGR male rats. Lastly, we hypothesized that IUGR male rats would perform poorer on tests of hippocampal function at P120. IUGR was induced by bilateral ligation of the uterine arteries in pregnant dams at embryonic day 19 (term is 21 days). Hippocampal Igf1 mRNA transcript levels and protein abundance were unchanged in IUGR male rats at P0, P21 or P120. At P0 and P120, IGF1r expression was increased in IUGR male rats. At P21, IGF1r expression was decreased in IUGR male rats. Increased IGF1r expression was associated with more histone 3 lysine 4 dimethylation (H3K4Me2) in the promoter region. In addition, IUGR male rats performed poorer on intermediate-term spatial working memory testing at P120. We speculate that altered IGF1r expression in the hippocampus of IUGR male rats may play a role in learning and memory dysfunction later in life.

Epigenetics and fetal adaptation to perinatal events: diversity through fidelity.

Perinatal insults, including fetal undernutrition and hypoxia, are associated with an increased susceptibility to several adult-onset metabolic disorders. These include cardiovascular disease, insulin resistance, and obesity. However, the mechanisms driving the long-term phenotypic consequences have only recently begun to be elucidated. A primary mechanism accounting for perinatal adaptation is the epigenetic modification of chromatin. In this context, epigenetic modifications to chromatin are thought to arise in response to a perinatal insult in an effort to modulate gene expression and maximize fetal survival. In this symposium report, we discuss epigenetics as a mechanism by which perinatal adaptations can be made by the developing fetus. We examine the benefits of using multiple in vivo models to understand the interrelation of signals that come together and result in perinatal adaptation. Epigenetic effects on IGF-1 arising from a perinatal insult are discussed, as are the difficulties and challenges associated with this complex field. In conclusion, epigenetics provides a means of modulating gene transcription, thus allowing fetal adaptation to a broad variety of conditions.

Uteroplacental insufficiency decreases p53 serine-15 phosphorylation in term IUGR rat lungs.

Intrauterine growth restriction (IUGR) increases the incidence of chronic lung disease (CLD). The molecular mechanisms responsible for IUGR-induced acute lung injury that predispose the IUGR infant to CLD are unknown. p53, a transcription factor, plays a pivotal role in determining cellular response to stress by affecting apoptosis, cell cycle regulation, and angiogenesis, processes required for thinning of lung mesenchyme. Because thickened lung mesenchyme is characteristic of CLD, we hypothesized that IUGR-induced changes in lung growth are associated with alterations in p53 expression and/or modification. We induced IUGR through bilateral uterine artery ligation of pregnant rats. Uteroplacental insufficiency significantly decreased serine-15-phosphorylated (serine-15P) p53, an active form of p53, in IUGR rat lung. Moreover, we found that decreased phosphorylation of lung p53 serine-15 localized to thickened distal air space mesenchyme. We also found that IUGR significantly decreased mRNA for targets downstream of p53, specifically, proapoptotic Bax and Apaf, as well as Gadd45, involved in growth arrest, and Tsp-1, involved in angiogenesis. Furthermore, we found that IUGR significantly increased mRNA for Bcl-2, an antiapoptotic gene downregulated by p53. We conclude that in IUGR rats, uteroplacental insufficiency induces decreased lung mesenchymal p53 serine-15P in association with distal lung mesenchymal thickening. We speculate that decreased p53 serine-15P in IUGR rat lungs alters lung phenotype, making the IUGR lung more susceptible to subsequent injury.

Uteroplacental insufficiency affects epigenetic determinants of chromatin structure in brains of neonatal and juvenile IUGR rats.

Intrauterine growth retardation (IUGR) increases the risk of neuroendocrine reprogramming. In the rat, IUGR leads to persistent changes in cerebral mRNA levels. This suggests lasting alterations in IUGR cerebral transcriptional regulation, which may result from changes in chromatin structure. Candidate nutritional triggers for these changes include altered cerebral zinc and one-carbon metabolite levels. We hypothesized that IUGR affects cerebral chromatin structure in neonatal and postnatal rat brains. Rats were rendered IUGR by bilateral uterine artery ligation; controls (Con) underwent sham surgery. At day of life 0 (d0), we measured cerebral DNA methylation, histone acetylation, expression of chromatin-affecting enzymes, and cerebral levels of one-carbon metabolites and zinc. At day of life 21 (d21), we measured cerebral DNA methylation and histone acetylation, as well as the caloric content of Con and IUGR rat breast milk. At d0, IUGR significantly decreased genome-wide and CpG island methylation, as well as increased histone 3 lysine 9 (H3/K9) and histone 3 lysine 14 (H3/K14) acetylation in the hippocampus and periventricular white matter, respectively. IUGR also decreased expression of the chromatin-affecting enzymes DNA methyltransferase 1 (DNMT1), methyl-CpG binding protein 2 (MeCP2), and histone deacetylase (HDAC)1 in association with increased cerebral levels of zinc. In d21 female IUGR rats, cerebral CpG DNA methylation remained lower, whereas H3/K9 and H3/K14 hyperacetylation persisted in hippocampus and white matter, respectively. In d21 male rats, IUGR decreased acetylation of H3/K9 and H3/K14 in these respective regions compared with controls. Despite these differences, caloric, fat, and protein content were similar in breast milk from Con and IUGR dams. We conclude that IUGR results in postnatal changes in cerebral chromatin structure and that these changes are sex specific.

GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst.

Mammalian preimplantation blastocysts exhibit insulin-stimulated glucose uptake despite the absence of the only known insulin-regulated transporter, GLUT4. We describe a previously unidentified member of the mammalian facilitative GLUT superfamily that exhibits approximately 20-25% identity with other murine facilitative GLUTs. Insulin induces a change in the intracellular localization of this protein, which translates into increased glucose uptake into the blastocyst, a process that is inhibited by antisense oligoprobes. Presence of this transporter may be necessary for successful blastocyst development, fuel metabolism, and subsequent implantation. Moreover, the existence of an alternative transporter may explain examples in other tissues of insulin-regulated glucose transport in the absence of GLUT4.

Effect of development and hypoxic-ischemia upon rabbit brain glucose transporter expression.

We have cloned and sequenced a full length rabbit GLUT 1 and partial rabbit GLUT 3 cDNAs. The derived rabbit GLUT 3 peptide revealed 84% homology to the mouse, 82% to the rat, human, dog, and sheep, and 69% to the chicken GLUT 3 peptides. Using Northern blot analysis, we investigated the tissue and brain cellular distribution of GLUT 1 and GLUT 3 expression. In addition, we examined the effect of development and hypoxic-ischemia upon brain GLUT 1 and GLUT 3 mRNA levels. While GLUT 1 mRNA was observed in most tissues, GLUT 3 was expressed predominantly in the brain, placenta, stomach, and lung with minor amounts in the heart, kidney and skeletal muscle. In the brain, both GLUT 1 and GLUT 3 were noted in neuron- and glial-enriched cultures. Both GLUT 1 and GLUT 3 mRNA levels demonstrated a similar developmental progression (p<0.05) secondary to post-transcriptional mechanisms. Further, while hypoxic-ischemia did not significantly affect brain GLUT 1 mRNA and protein, it altered GLUT 3 mRNA levels in a region-specific manner, with a three-fold increase in the cerebral cortex, a two-fold increase in the hippocampus, and a 50% increase in the caudate nucleus (p<0.05). We conclude, that the rabbit GLUT 3 peptide sequence exhibits 82-84% homology to that of other species in the coding region with a 62-89% sequence identity in the 3'-untranslated region. The tissue-specific expression of rabbit GLUT 3 mimics that of the human closely. Postnatal development and hypoxic-ischemia with reperfusion injury cause an increase in brain GLUT 3 expression, as a response to synaptogenesis and substrate deprivation, respectively.

Developmental regulation of genes mediating murine brain glucose uptake.

We examined the molecular mechanisms that mediate the developmental increase in murine whole brain 2-deoxyglucose uptake. Northern and Western blot analyses revealed an age-dependent increase in brain GLUT-1 (endothelial cell and glial) and GLUT-3 (neuronal) membrane-spanning facilitative glucose transporter mRNA and protein concentrations. Nuclear run-on experiments revealed that these developmental changes in GLUT-1 and -3 were regulated posttranscriptionally. In contrast, the mRNA and protein levels of the mitochondrially bound glucose phosphorylating hexokinase I enzyme were unaltered. However, hexokinase I enzyme activity increased in an age-dependent manner suggestive of a posttranslational modification that is necessary for enzymatic activation. Together, the postnatal increase in GLUT-1 and -3 concentrations and hexokinase I enzymatic activity led to a parallel increase in murine brain 2-deoxyglucose uptake. Whereas the molecular mechanisms regulating the increase in the three different gene products may vary, the age-dependent increase of all three constituents appears essential for meeting the increasing demand of the maturing brain to fuel the processes of cellular growth, differentiation, and neurotransmission.

Severe position effects imposed on a 1 kb mouse whey acidic protein gene promoter are overcome by heterologous matrix attachment regions.

Matrix attachment regions (MARs) have been shown to participate in the insulation of transcription elements from surrounding chromatin in tissue culture cells and transgenic mice. A whey acidic protein (WAP) transgene containing 1 kb promoter sequence was active in mammary tissue from 1 out of 17 lines of mice, demonstrating that the transcription elements were highly susceptible to position effects. To test whether MARs could insulate this WAP gene promoter and thereby restore transcription, we ligated MARs from the chicken lysozyme gene to the WAP transgene. Seven of the nine lines generated exhibited WAP transgene activity, expression was confined to mammary tissue, and correct regulation was observed in three of the four lines analyzed. This study provides strong additional evidence that the MAR fragments from the chicken lysozyme gene have the capacity to insulate transgenes from severe position effects.

Mammary epithelial cells undergo secretory differentiation in cycling virgins but require pregnancy for the establishment of terminal differentiation.

Postnatal development of the mammary gland begins during puberty with ductal proliferation and is completed at delivery with the appearance of secretory alveolar structures. Using endogenous milk protein genes and a WAP-lacZ reporter transgene, we show that the differentiation of alveolar cells is initiated in virgin mice in estrus in a limited number of cells. With the onset of pregnancy, the number of expressing cells and the cellular expression levels increase until full activity is reached at lactation. Milk protein genes are activated in a defined temporal sequence. WDNM1 and beta-casein are expressed early in pregnancy and increase during alveolar proliferation. WAP (whey acidic protein) and alpha-lactalbumin are expressed later near the end of gestation, which is characterized by terminal differentiation of the mammary secretory phenotype. By in situ hybridization, we have established evidence for asynchrony in milk protein gene expression among alveolar cells showing large variations in the intensity of hybridization among adjacent cells. The asynchrony of maturation of epithelial cells within a given alveolus suggests that the genetic program leading to terminal differentiation is subject to local modulation. It is likely that these signals are manifest through various pathways including growth factors, the extracellular matrix or gene products specific to terminal differentiation such as WAP. We extended our analyses to WAP/WAP transgenic mice in which WAP is synthesized precociously and functional differentiation of alveolar cells is impaired. We found an altered expression pattern of milk protein genes, with a strong reduction of alpha-lactalbumin RNA. We conclude that the early production of WAP in WAP/WAP mammary glands disrupts the timing of gene activation leading to a premature termination of the differentiative program.

An Ets site in the whey acidic protein gene promoter mediates transcriptional activation in the mammary gland of pregnant mice but is dispensable during lactation.

The whey acidic protein (WAP) gene is specifically expressed in mammary tissue, and its transcription is induced several thousand-fold during pregnancy and remains high throughout lactation. A purine-rich sequence (PRS) located around -110 of the WAP gene promoter is conserved between mice, rats, and rabbits, suggesting that it features a regulatory element. This PRS contains an invariant GGAA/T core motif characteristic of the binding site for Ets transcription factors. Electromobility shift assays demonstrate that Ets1 binds specifically to the PRS. Experiments in transgenic mice further demonstrate that this PRS/Ets site plays a critical role in the activation of WAP transgenes during pregnancy, but that its presence is not required for high expression throughout lactation. Transgenes with an intact PRS/Ets site are expressed at high levels at day 13 of pregnancy, with little further increase during late pregnancy and lactation. In contrast, WAP transgenes with a mutation in the PRS/Ets site, which abrogates the binding of Ets1, are not expressed at midpregnancy, but their transcriptional activity is not affected during lactation. These results demonstrate that Ets-signaling pathways can function as stage-specific transcriptional activators of milk protein genes in the developing mammary gland. In addition, this work extends earlier findings that gene activation during pregnancy and lactation is mediated, in part, by different mechanisms.

Ectopic TGF beta 1 expression in the secretory mammary epithelium induces early senescence of the epithelial stem cell population.

An important feature of the mammary gland is the regenerative capacity of its epithelium which is demonstrated upon successive cycles of lactation and involution. Pregnant mice expressing a whey-acidic protein (WAP) promoter-driven transforming growth factor-beta 1 (TGF beta 1) cDNA are unable either to generate a secretory mammary epithelium or to lactate. Here we investigate whether ectopic TGF beta 1 induces this phenotype by affecting the transgenic epithelium directly or in trans. Reciprocal transplantation of mammary tissue between normal and transgenic hosts resulted in the development of the respective phenotypes of the transplants within the same mammary fat pad. When isolated mammary epithelial cells from both were mixed before implantation so that transgenic and normal epithelium would develop together more proximately, both phenotypes were simultaneously observed in the resultant chimeric mammary outgrowths. Since no trans effect was detectable, we hypothesize that early expression of the transgene results in compromised lobular progenitor cells through an intracrine mechanism. Consistent with this posit, WAP promoter-driven protein expression was detected in individual cells of the subtending ducts of immature females at estrus. Transplantation of WAP-TGF beta 1 mammary gland into nonpregnant hosts revealed that transgenic implants, even those from young postpubertal virgin females, had a diminished ability to repopulate epithelium-free mammary fat pads. Accordingly, the ectopic expression of WAP-TGF beta 1 not only impairs lobular progenitors, but also promotes an early senescence of the regenerative capacity of the mammary ductal epithelium. This leads us to propose that mammary epithelial stem cells give rise to two functionally distinct progenitor cells in the mammary gland epithelium: one capable of producing daughters committed to ductal formation, the other capable only of producing daughters committed to lobular function.

Expression of genomic and cDNA transgenes after co-integration in transgenic mice.

In general, genomic transgenes are expressed efficiently in mice, while their cDNA-based transgenes are frequently silent. Clark et al. (1992) have shown that silent cDNA transgenes under the control of the sheep beta-lactoglobulin promoter can be activated after co-injecting them with a genomic sheep beta-lactoglobulin transgene. We have tested the general utility of this concept using mouse whey acidic protein (WAP) transgenes. Here we show that WAP cDNA transgenes are virtually silent in transgenic mice. In contrast, WAP transgenes containing all introns are expressed in approximately 50% of the lines at levels ranging from 1% to more than 100% of the endogenous RNA (McKnight et al., 1992). When a WAP-genomic transgene was co-injected with a WAP-cDNA, basal activation of the cDNA was possible. However, the activity of the WAP-cDNA transgene did not exceed 1% of the WAP-genomic transgene. This suggests that a permissive integration site capable of supporting basal level transcription can be established, but further events are required for full activation of the transgene.

Regulation of human protein C gene expression by the mouse WAP promoter.

A 4.1 kb mouse whey acidic protein (mWAP) promoter was cloned from a C57BL/6 cosmid library. The tissue-specific and developmental pattern of expression of a hybrid gene comprised of the mWAP promoter fragment and the human protein C (HPC) gene was analysed in transgenic mice. The corresponding RNA was detected mainly in the mammary gland, with 'leakage' of expression in the salivary gland and kidney. The developmental pattern of transgene expression differed from that of the endogenous WAP gene. In particular, recombinant HPC (rHPC) transcripts were detected earlier in pregnancy than WAP RNA, with no significant increase during lactation. This indicates that regulatory elements responsible for developmental regulation are located outside the 4.1 kb mWAP gene promoter fragment, or if present, may be subject to position effects. Precocious expression of the transgene did not compromise the health or nursing abilities of transgenic females. Expression of rHPC affected the appearance of the mammary alveoli and alveolar epithelial cells in lactating transgenic mice. The alveoli were less distended and alveolar epithelial cells appeared cuboidal with centrally positioned nuclei. We suggest that the inefficient intracellular processing of rHPC can alter the histological appearance of alveolar epithelial cells in the transgenic mammary gland.

Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. We have tested whether MAR sequences from the chicken lysozyme locus, the so-called A-elements, can confer position-independent regulation to a whey acidic protein (WAP) transgene in mammary tissue of mice. In the absence of MARs, expression of WAP transgenes was observed in 50% of the lines, and regulation during pregnancy, during lactation, and upon hormonal induction did not mimic that of the endogenous WAP gene and varied with the integration site. In contrast, all 11 lines in which WAP transgenes were juxtaposed to MAR elements showed expression. Accurate position-independent hormonal and developmental regulation was seen in four out of the five lines analyzed. These results indicate that MARs can establish independent genetic domains in transgenic mice.

Production of the mouse whey acidic protein in transgenic pigs during lactation.

The mouse whey acidic protein (WAP) gene was introduced into the genome of pigs and its expression was analyzed in the mammary gland. Mouse WAP was detected in milk of lactating females from five lines at levels between .5 and 1.5 g/liter, thereby representing as much as 2% of the total milk proteins. The corresponding mRNA was expressed in mammary tissue at levels similar to those of pig beta-lactoglobulin and beta-casein. The pattern of WAP secretion in three pigs over a period of 6 wk was quantitatively similar to that of pig beta-lactoglobulin. From the eight transgenic pigs analyzed, three successfully completed one lactational period, but five pigs stopped lactating a few days after parturition. Our results show that it is possible to produce large quantities of a foreign protein in milk of pigs over a full lactational period. However, expression of WAP can compromise the mammary gland and render it nonfunctional.

High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine.

The whey acidic protein (WAP) is a major milk protein in mice, rats, and rabbits but has not been found in milk of livestock including swine. To determine whether mammary gland regulatory elements from the WAP gene function across species boundaries and whether it is possible to qualitatively alter milk protein composition, we introduced the mouse WAP gene into the genome of swine. Three lines of transgenic swine were analyzed, and mouse WAP was detected in milk from all lactating females at concentrations of about 1 g/liter; these levels are similar to those found in mouse milk. Expression of the corresponding RNA was specific to the mammary gland. Our results suggest that the molecular basis of mammary-specific gene expression is conserved between swine and mouse. In addition the WAP gene must share, with other milk protein genes, elements that target gene expression to the mammary gland. Mouse WAP accounted for about 3% of the total milk proteins in transgenic pigs, thus demonstrating that it is possible to produce high levels of a foreign protein in milk of farm animals. that it is possible to produce high levels of a foreign protein in

Cloning and sequencing of a complementary deoxyribonucleic acid coding for a bovine alpha s1-casein A from mammary tissue of a homozygous B variant cow.

A cDNA clone for bovine alpha s1-casein variant A was isolated from a mammary gland cDNA library using a synthetic degenerate oligonucleotide probe. The largest Pst I insert containing an EcoR I site was sequenced. It contained 1090 base pairs, 47 in the 5' noncoding region, 603 in the coding region and 440 in the 3' noncoding region. The nucleotide sequence was compared with three published cDNA sequences for alpha s1-casein variant B. The most obvious difference was the absence of the 39 bases encoding the 13 amino acids that are present in the B variant but absent from the A variant. In addition, five other single base positions differed within individual codons among the four sequences at the third base for each codon, but this did not change the amino acids encoded. There were, however, a number of differences found in the 3' noncoding region. The isolated cDNA was subjected to site-directed mutagenesis to replace a Val-Ile dipeptide with Phe-Phe to increase the chymosin sensitivity of the protein. When the milk proteins from mammary gland tissue extracts were typed, the alpha s1-casein A gene product was not detected.