Mesenchyme-derived IGF2 is a major paracrine regulator of pancreatic growth and function.

The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Imprinted genes, which are expressed in a parent-of-origin-specific manner, are known to have important roles in development, growth and metabolism. However, our knowledge regarding their roles in the control of pancreatic growth and function remains limited. Here we show that many imprinted genes are highly expressed in pancreatic mesenchyme-derived cells and explore the role of the paternally-expressed insulin-like growth factor 2 (Igf2) gene in mesenchymal and epithelial pancreatic lineages using a newly developed conditional Igf2 mouse model. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of any discernible growth or functional phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Additionally, increased IGF2 levels specifically in the mesenchyme, through conditional Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Furthermore, ex-vivo exposure of primary acinar cells to exogenous IGF2 activates AKT, a key signalling node, and increases their number and amylase production. Based on these findings, we propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.

Sex-specific regulation of stress-induced fetal glucocorticoid surge by the mouse placenta.

Antenatal stress increases the prevalence of diseases in later life, which shows a strong sex-specific effect. However, the underlying mechanisms remain unknown. Maternal glucocorticoids can be elevated by stress and are potential candidates to mediate the effects of stress on the offspring sex-specifically. A comprehensive evaluation of dynamic maternal and placental mechanisms modulating fetal glucocorticoid exposure upon maternal stress was long overdue. Here, we addressed this gap in knowledge by investigating sex-specific responses to midgestational stress in mice. We observed increased levels of maternal corticosterone, the main glucocorticoid in rodents, along with higher corticosteroid-binding globulin levels at midgestation in C57Bl/6 dams exposed to sound stress. This resulted in elevated corticosterone in female fetuses, whereas male offspring were unaffected. We identified that increased placental expression of the glucocorticoid-inactivating enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2; Hsd11b2 gene) and ATP-binding cassette transporters, which mediate glucocorticoid efflux toward maternal circulation, protect male offspring from maternal glucocorticoid surges. We generated mice with an Hsd11b2 placental-specific disruption (Hsd11b2PKO) and observed moderately elevated corticosterone levels in offspring, along with increased body weight. Subsequently, we assessed downstream glucocorticoid receptors and observed a sex-specific differential modulation of placental Tsc22d3 expression, which encodes the glucocorticoid-induced leucine zipper protein in response to stress. Taken together, our observations highlight the existence of unique and well-orchestrated mechanisms that control glucocorticoid transfer, exposure, and metabolism in the mouse placenta, pinpointing toward the existence of sex-specific fetal glucocorticoid exposure windows during gestation in mice.

Maternal and fetal genomes interplay through phosphoinositol 3-kinase(PI3K)-p110α signaling to modify placental resource allocation.

Pregnancy success and life-long health depend on a cooperative interaction between the mother and the fetus in the allocation of resources. As the site of materno-fetal nutrient transfer, the placenta is central to this interplay; however, the relative importance of the maternal versus fetal genotypes in modifying the allocation of resources to the fetus is unknown. Using genetic inactivation of the growth and metabolism regulator, Pik3ca (encoding PIK3CA also known as p110α, α/+), we examined the interplay between the maternal genome and the fetal genome on placental phenotype in litters of mixed genotype generated through reciprocal crosses of WT and α/+ mice. We demonstrate that placental growth and structure were impaired and associated with reduced growth of α/+ fetuses. Despite its defective development, the α/+ placenta adapted functionally to increase the supply of maternal glucose and amino acid to the fetus. The specific nature of these changes, however, depended on whether the mother was α/+ or WT and related to alterations in endocrine and metabolic profile induced by maternal p110α deficiency. Our findings thus show that the maternal genotype and environment programs placental growth and function and identify the placenta as critical in integrating both intrinsic and extrinsic signals governing materno-fetal resource allocation.

Intergenerational epigenetic inheritance in models of developmental programming of adult disease.

It is now well established that the environment to which we are exposed during fetal and neonatal life can have a long-term impact on our health. This has been termed the developmental origins of health and disease. Factors known to have such programming effects include intrauterine nutrient availability (determined by maternal nutrition and placental function), endocrine disruptors, toxins and infectious agents. Epigenetic processes have emerged as a key mechanism by which the early environment can permanently influence cell function and metabolism after multiple rounds of cell division. More recently it has been suggested that programmed effects can be observed beyond the first generation and that therefore epigenetic mechanisms could form the basis of transmission of phenotype from parent to child to grandchild and beyond. Here we review the evidence for such processes.

Placental phenotype and the insulin-like growth factors: resource allocation to fetal growth.

The placenta is the main determinant of fetal growth and development in utero. It supplies all the nutrients and oxygen required for fetal growth and secretes hormones that facilitate maternal allocation of nutrients to the fetus. Furthermore, the placenta responds to nutritional and metabolic signals in the mother by altering its structural and functional phenotype, which can lead to changes in maternal resource allocation to the fetus. The molecular mechanisms by which the placenta senses and responds to environmental cues are poorly understood. This review discusses the role of the insulin-like growth factors (IGFs) in controlling placental resource allocation to fetal growth, particularly in response to adverse gestational environments. In particular, it assesses the impact of the IGFs and their signalling machinery on placental morphogenesis, substrate transport and hormone secretion, primarily in the laboratory species, although it draws on data from human and other species where relevant. It also considers the role of the IGFs as environmental signals in linking resource availability to fetal growth through changes in the morphological and functional phenotype of the placenta. As altered fetal growth is associated with increased perinatal morbidity and mortality and a greater risk of developing adult-onset diseases in later life, understanding the role of IGFs during pregnancy in regulating placental resource allocation to fetal growth is important for identifying the mechanisms underlying the developmental programming of offspring phenotype by suboptimal intrauterine growth.

Ageing is associated with molecular signatures of inflammation and type 2 diabetes in rat pancreatic islets.

AIMS/HYPOTHESIS: Ageing is a major risk factor for development of metabolic diseases such as type 2 diabetes. Identification of the mechanisms underlying this association could help to elucidate the relationship between age-associated progressive loss of metabolic health and development of type 2 diabetes. We aimed to determine molecular signatures during ageing in the endocrine pancreas. METHODS: Global gene transcription was measured in pancreatic islets isolated from young and old rats by Ilumina BeadChip arrays. Promoter DNA methylation was measured by Sequenom MassArray in 46 genes that showed differential expression with age, and correlations with expression were established. Alterations in morphological and cellular processes with age were determined by immunohistochemical methods. RESULTS: Age-related changes in gene expression were found at 623 loci (>1.5-fold, false discovery rate [FDR] <5%), with a significant (FDR < 0.05) enrichment in genes previously implicated in islet-cell function (Enpp1, Abcc8), type 2 diabetes (Tspan8, Kcnq1), inflammatory processes (Cxcl9, Il33) and extracellular matrix organisation (Col3a1, Dpt). Age-associated transcriptional differences negatively correlated with promoter DNA methylation at several loci related to inflammation, glucose homeostasis, cell proliferation and cell-matrix interactions (Il33, Cxcl9, Gpr119, Fbp2, Col3a1, Dpt, Spp1). CONCLUSIONS/INTERPRETATION: Our findings suggest that a significant proportion of pancreatic islets develop a low-grade 'chronic' inflammatory status with ageing and this may trigger altered functional plasticity. Furthermore, we identified changes in expression of genes previously linked to type 2 diabetes and associated changes in DNA methylation that could explain their age-associated dysregulation. These findings provide new insights into key (epi)genetic signatures of the ageing process in islets.

Maternal diet amplifies the hepatic aging trajectory of Cidea in male mice and leads to the development of fatty liver.

The importance of the early environment on long-term heath and life span is well documented. However, the molecular mechanisms mediating these effects remain poorly understood. Male offspring from a maternal protein restriction model, in which animals are exposed to a low-protein diet while in utero and then are cross-fostered to normally fed dams, demonstrate low birth weight, catch-up growth, and reduced life span (recuperated offspring). In the current study, we used microarray analysis to identify hepatic genes that changed with age. Cell death-inducing DNA fragmentation factor, α subunit-like effector A (Cidea), a transcriptional coactivator that has been implicated in lipid accumulation demonstrated one of the largest age-associated increases in expression (200-fold, P<0.001). This increase was exaggerated ∼3-fold in recuperated offspring. These demonstrated increased hepatic lipid accumulation, higher levels of transcription factors important in lipid regulation, and greater oxidative stress. In vitro analysis revealed that Cidea expression was regulated by oxidative stress and DNA methylation. These findings suggest that maternal diet modulates the age-associated changes in Cidea expression through several mechanisms. This expression affects hepatic lipid metabolism in these animals and thus provides a mechanism by which maternal diet can contribute to the metabolic health and ultimately the life span of the offspring.

Impact on offspring methylation patterns of maternal gestational diabetes mellitus and intrauterine growth restraint suggest common genes and pathways linked to subsequent type 2 diabetes risk.

Size at birth, postnatal weight gain, and adult risk for type 2 diabetes may reflect environmental exposures during developmental plasticity and may be mediated by epigenetics. Both low birth weight (BW), as a marker of fetal growth restraint, and high birth weight (BW), especially after gestational diabetes mellitus (GDM), have been linked to increased risk of adult type 2 diabetes. We assessed DNA methylation patterns using a bead chip in cord blood samples from infants of mothers with GDM (group 1) and infants with prenatal growth restraint indicated by rapid postnatal catch-up growth (group 2), compared with infants with normal postnatal growth (group 3). Seventy-five CpG loci were differentially methylated in groups 1 and 2 compared with the controls (group 3), representing 72 genes, many relevant to growth and diabetes. In replication studies using similar methodology, many of these differentially methylated regions were associated with levels of maternal glucose exposure below that defined by GDM [the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study] or were identified as changes observed after randomized periconceptional nutritional supplementation in a Gambian cohort characterized by maternal deprivation. These studies provide support for the concept that similar epigenetic modifications may underpin different prenatal exposures and potentially increase long-term risk for diseases such as type 2 diabetes.

Periconceptional maternal micronutrient supplementation is associated with widespread gender related changes in the epigenome: a study of a unique resource in the Gambia.

In addition to the genetic constitution inherited by an organism, the developmental trajectory and resulting mature phenotype are also determined by mechanisms acting during critical windows in early life that influence and establish stable patterns of gene expression. This is the crux of the developmental origins of health and disease hypothesis that suggests undernutrition during gestation and infancy predisposes to ill health in later life. The hypothesis that periconceptional maternal micronutrient supplementation might affect fetal genome-wide methylation within gene promoters was explored in cord blood samples from offspring of Gambian women enrolled into a unique randomized, double blind controlled trial. Significant changes in the epigenome in cord blood DNA samples were further explored in a subset of offspring at 9 months. Gender-specific changes related to periconceptional nutritional supplementation were identified in cord blood DNA samples, some of which showed persistent changes in infant blood DNA samples. Significant effects of periconceptional micronutrient supplementation were also observed in postnatal samples which were not evident in cord blood. In this Gambian population, the increased death rate of individuals born in nutritionally poor seasons has been related to infection and it is of interest that we identified differential methylation at genes associated with defence against infection and immune response. Although the sample size was relatively small, these pilot data suggest that periconceptional nutrition in humans is an important determinant of newborn whole genome methylation patterns but may also influence postnatal developmental patterns of gene promoter methylation linking early with disease risk.

An obesogenic diet during mouse pregnancy modifies maternal nutrient partitioning and the fetal growth trajectory.

In developed societies, high-sugar and high-fat (HSHF) diets are now the norm and are increasing the rates of maternal obesity during pregnancy. In pregnant rodents, these diets lead to cardiovascular and metabolic dysfunction in their adult offspring, but the intrauterine mechanisms involved remain unknown. This study shows that, relative to standard chow, HSHF feeding throughout mouse pregnancy increases maternal adiposity (+30%, P<0.05) and reduces fetoplacental growth at d 16 (-10%, P<0.001). At d 19, however, HSHF diet group pup weight had normalized, despite the HSHF diet group placenta remaining small and morphologically compromised. This altered fetal growth trajectory was associated with enhanced placental glucose and amino acid transfer (+35%, P<0.001) and expression of their transporters (+40%, P<0.024). HSHF feeding also up-regulated placental expression of fatty acid transporter protein, metabolic signaling pathways (phosphoinositol 3-kinase and mitogen-activated protein kinase), and several growth regulatory imprinted genes (Igf2, Dlk1, Snrpn, Grb10, and H19) independently of changes in DNA methylation. Obesogenic diets during pregnancy, therefore, alter maternal nutrient partitioning, partly through changes in the placental phenotype, which helps to meet fetal nutrient demands for growth near term. However, by altering provision of specific nutrients, dietary-induced placental adaptations have important roles in programming development with health implications for the offspring in later life.

Developmental and environmental epigenetic programming of the endocrine pancreas: consequences for type 2 diabetes.

The development of the endocrine pancreas is controlled by a hierarchical network of transcriptional regulators. It is increasingly evident that this requires a tightly interconnected epigenetic "programme" to drive endocrine cell differentiation and maintain islet function. Epigenetic regulators such as DNA and histone-modifying enzymes are now known to contribute to determination of pancreatic cell lineage, maintenance of cellular differentiation states, and normal functioning of adult pancreatic endocrine cells. Persistent effects of an early suboptimal environment, known to increase risk of type 2 diabetes in later life, can alter the epigenetic control of transcriptional master regulators, such as Hnf4a and Pdx1. Recent genome-wide analyses also suggest that an altered epigenetic landscape is associated with the ß cell failure observed in type 2 diabetes and aging. At the cellular level, epigenetic mechanisms may provide a mechanistic link between energy metabolism and stable patterns of gene expression. Key energy metabolites influence the activity of epigenetic regulators, which in turn alter transcription to maintain cellular homeostasis. The challenge is now to understand the detailed molecular mechanisms that underlie these diverse roles of epigenetics, and the extent to which they contribute to the pathogenesis of type 2 diabetes. In-depth understanding of the developmental and environmental epigenetic programming of the endocrine pancreas has the potential to lead to novel therapeutic approaches in diabetes.

Maternal diet and aging alter the epigenetic control of a promoter-enhancer interaction at the Hnf4a gene in rat pancreatic islets.

Environmental factors interact with the genome throughout life to determine gene expression and, consequently, tissue function and disease risk. One such factor that is known to play an important role in determining long-term metabolic health is diet during critical periods of development. Epigenetic regulation of gene expression has been implicated in mediating these programming effects of early diet. The precise epigenetic mechanisms that underlie these effects remain largely unknown. Here, we show that the transcription factor Hnf4a, which has been implicated in the etiology of type 2 diabetes (T2D), is epigenetically regulated by maternal diet and aging in rat islets. Transcriptional activity of Hnf4a in islets is restricted to the distal P2 promoter through its open chromatin configuration and an islet-specific interaction between the P2 promoter and a downstream enhancer. Exposure to suboptimal nutrition during early development leads to epigenetic silencing at the enhancer region, which weakens the P2 promoter-enhancer interaction and results in a permanent reduction in Hnf4a expression. Aging leads to progressive epigenetic silencing of the entire Hnf4a locus in islets, an effect that is more pronounced in rats exposed to a poor maternal diet. Our findings provide evidence for environmentally induced epigenetic changes at the Hnf4a enhancer that alter its interaction with the P2 promoter, and consequently determine T2D risk. We therefore propose that environmentally induced changes in promoter-enhancer interactions represent a fundamental epigenetic mechanism by which nutrition and aging can influence long-term health.

DNA methylation profiling at imprinted loci after periconceptional micronutrient supplementation in humans: results of a pilot randomized controlled trial.

Intrauterine exposures mediated by maternal diet may affect risk of cardiovascular disease, obesity, and type 2 diabetes. Recent evidence, primarily from animal studies and observational data in humans, suggests that the epigenome can be altered by maternal diet during the periconceptional period and that these programming events may underlie later disease risk. A randomized controlled trial of periconceptional micronutrient supplementation in The Gambia, where seasonal nutritional variations affect fetal growth and postnatal outcomes, provided a unique opportunity to test this hypothesis. Specifically, we targeted imprinted genes, which play important roles in allocation of maternal resources while being epigenetically regulated. DNA methylation at 12 differentially methylated regions (DMRs) was analyzed in cord blood samples from 58 offspring of women participating in a double-blind randomized-controlled trial of pre- and periconceptional micronutrient supplementation (including folate, zinc, and vitamins A, B, C, and D). We observed sex-specific effects of micronutrient supplementation, reducing methylation levels at two of the DMRs analyzed, IGF2R in girls and GTL2-2 in boys. This pilot study is the first to analyze DNA methylation in the context of a randomized controlled trial, and it provides suggestive evidence that periconceptional maternal nutrition alters offspring methylation at imprinted loci.

Neuronal activity drives IGF2 expression from pericytes to form long-term memory.

Investigations of memory mechanisms have been, thus far, neuron centric, despite the brain comprising diverse cell types. Using rats and mice, we assessed the cell-type-specific contribution of hippocampal insulin-like growth factor 2 (IGF2), a polypeptide regulated by learning and required for long-term memory formation. The highest level of hippocampal IGF2 was detected in pericytes, the multi-functional mural cells of the microvessels that regulate blood flow, vessel formation, the blood-brain barrier, and immune cell entry into the central nervous system. Learning significantly increased pericytic Igf2 expression in the hippocampus, particularly in the highly vascularized stratum lacunosum moleculare and stratum moleculare layers of the dentate gyrus. Igf2 increases required neuronal activity. Regulated hippocampal Igf2 knockout in pericytes, but not in fibroblasts or neurons, impaired long-term memories and blunted the learning-dependent increase of neuronal immediate early genes (IEGs). Thus, neuronal activity-driven signaling from pericytes to neurons via IGF2 is essential for long-term memory.

Fetal manipulation of maternal metabolism is a critical function of the imprinted Igf2 gene.

Maternal-offspring interactions in mammals involve both cooperation and conflict. The fetus has evolved ways to manipulate maternal physiology to enhance placental nutrient transfer, but the mechanisms involved remain unclear. The imprinted Igf2 gene is highly expressed in murine placental endocrine cells. Here, we show that Igf2 deletion in these cells impairs placental endocrine signaling to the mother, without affecting placental morphology. Igf2 controls placental hormone production, including prolactins, and is crucial to establish pregnancy-related insulin resistance and to partition nutrients to the fetus. Consequently, fetuses lacking placental endocrine Igf2 are growth restricted and hypoglycemic. Mechanistically, Igf2 controls protein synthesis and cellular energy homeostasis, actions dependent on the placental endocrine cell type. Igf2 loss also has additional long-lasting effects on offspring metabolism in adulthood. Our study provides compelling evidence for an intrinsic fetal manipulation system operating in placenta that modifies maternal metabolism and fetal resource allocation, with long-term consequences for offspring metabolic health.

Developmental adaptations to increased fetal nutrient demand in mouse genetic models of Igf2-mediated overgrowth.

The healthy development of the fetus depends on an optimal balance between fetal genetic drive for growth and the maternal ability to provide nutrients through the placenta. Nothing is known about fetal-placental signaling in response to increased fetal demand in the situation of overgrowth. Here, we examined this question using the H19(Δ13) mouse model, shown previously to result in elevated levels of Igf2. Fetal and placental weights in H19(Δ13) were increased by 23% and 45%, respectively, at E19, when compared with wild-type mice. Unexpectedly, we found that disproportionately large H19(Δ13) placentas transport 20-35% less (per gram placenta) glucose and system A amino acids and have similar reductions in passive permeability, despite a significantly greater surface area for nutrient exchange and theoretical diffusion capacity compared with wild-type mice. Expression of key transporter genes Slc2a3 and Slc38a4 was reduced by ∼20%. Decreasing the overgrowth of the H19(Δ13) placenta by genetically reducing levels of Igf2P0 resulted in up-regulation of system A activity and maintenance of fetal overgrowth. Our results provide direct evidence that large placentas can modify their nutrient transfer capacity to regulate fetal nutrient acquisition. Our findings are indicative of fetal-placental signaling mechanisms that limit total demand for maternal nutrients.

Placental adaptations to the maternal-fetal environment: implications for fetal growth and developmental programming.

The placenta is a transient organ found in eutherian mammals that evolved primarily to provide nutrients for the developing fetus. The placenta exchanges a wide array of nutrients, endocrine signals, cytokines and growth factors with the mother and the fetus, thereby regulating intrauterine development. Recent studies show that the placenta is not just a passive organ mediating maternal-fetal exchange. It can adapt its capacity to supply nutrients in response to intrinsic and extrinsic variations in the maternal-fetal environment. These dynamic adaptations are thought to occur to maximize fetal growth and viability at birth in the prevailing conditions in utero. However, some of these adaptations may also affect the development of individual fetal tissues, with patho-physiological consequences long after birth. Here, this review summarizes current knowledge on the causes, possible mechanisms and consequences of placental adaptive responses, with a focus on the regulation of transporter-mediated processes for nutrients. This review also highlights the emerging roles that imprinted genes and epigenetic mechanisms of gene regulation may play in placental adaptations to the maternal-fetal environment.

Protocol to isolate and culture primary mouse feto-placental endothelial cells.

In the mouse, feto-placental endothelial cells (FPEC) line the inner surface of the feto-placental blood vessels located within placental labyrinthine zone and play critical roles in placental development and function. Here, we present a detailed protocol for isolation and culture of primary mouse FPEC, as well as two complementary methods (immunohistochemistry staining and flow cytometry analysis) to assess their purity. These cells are suitable for downstream ex vivo studies to investigate their functional properties, both in normal and pathological contexts. For complete details on the use and execution of this protocol, please refer to Sandovici et al. (2022).

Sex differences in the intergenerational inheritance of metabolic traits.

Strong evidence suggests that early-life exposures to suboptimal environmental factors, including those in utero, influence our long-term metabolic health. This has been termed developmental programming. Mounting evidence suggests that the growth and metabolism of male and female fetuses differ. Therefore, sexual dimorphism in response to pre-conception or early-life exposures could contribute to known sex differences in susceptibility to poor metabolic health in adulthood. However, until recently, many studies, especially those in animal models, focused on a single sex, or, often in the case of studies performed during intrauterine development, did not report the sex of the animal at all. In this review, we (a) summarize the evidence that male and females respond differently to a suboptimal pre-conceptional or in utero environment, (b) explore the potential biological mechanisms that underlie these differences and (c) review the consequences of these differences for long-term metabolic health, including that of subsequent generations.

The imprinted Igf2-Igf2r axis is critical for matching placental microvasculature expansion to fetal growth.

In all eutherian mammals, growth of the fetus is dependent upon a functional placenta, but whether and how the latter adapts to putative fetal signals is currently unknown. Here, we demonstrate, through fetal, endothelial, hematopoietic, and trophoblast-specific genetic manipulations in the mouse, that endothelial and fetus-derived IGF2 is required for the continuous expansion of the feto-placental microvasculature in late pregnancy. The angiocrine effects of IGF2 on placental microvasculature expansion are mediated, in part, through IGF2R and angiopoietin-Tie2/TEK signaling. Additionally, IGF2 exerts IGF2R-ERK1/2-dependent pro-proliferative and angiogenic effects on primary feto-placental endothelial cells ex vivo. Endothelial and fetus-derived IGF2 also plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. Thus, our study reveals a direct role for the imprinted Igf2-Igf2r axis on matching placental development to fetal growth and establishes the principle that hormone-like signals from the fetus play important roles in controlling placental microvasculature and trophoblast morphogenesis.