Prenatal programming of postnatal obesity: fetal nutrition and the regulation of leptin synthesis and secretion before birth

Exposure to either an increased or decreased level of intrauterine nutrition can result in an increase in adiposity and in circulating leptin concentrations in later life. In animals such as the sheep and pig in which fat is deposited before birth, leptin is synthesised in fetal adipose tissue and is present in the fetal circulation throughout late gestation. In the sheep a moderate increase or decrease in the level of maternal nutrition does not alter fetal plasma leptin concentrations, but there is evidence that chronic fetal hyperglycaemia and hyperinsulinaemia increase fetal fat mass and leptin synthesis within fetal fat depots. Importantly, there is a positive relationship between the relative mass of the ‘unilocular’ component of fetal perirenal and interscapular adipose tissue and circulating fetal leptin concentrations in the sheep. Thus, as in the neonate and adult, circulating leptin concentrations may be a signal of fat mass in fetal life. There is also evidence that leptin can act to regulate the lipid storage, leptin synthetic capacity and potential thermogenic functions of fat before birth. Thus, leptin may act as a signal of energy supply and have a ‘lipostatic’ role before birth. Future studies are clearly required to determine whether the intrauterine and early postnatal nutrient environment programme the endocrine feedback loop between adipose tissue and the central and peripheral neuroendocrine systems that regulate energy balance, resulting in an enhanced risk of obesity in adult life.I. C. McMillen, B. S. Muhlhausler, J. A. Duffield and B. S. J. Yue

Early restriction of placental growth results in placental structural and gene expression changes in late gestation independent of fetal hypoxemia

Placental restriction and insufficiency are associated with altered patterns of placental growth, morphology, substrate transport capacity, growth factor expression, and glucocorticoid exposure. We have used a pregnant sheep model in which the intrauterine environment has been perturbed by uterine carunclectomy (Cx). This procedure results in early restriction of placental growth and either the development of chronic fetal hypoxemia (PaO₂≤17 mmHg) in late gestation or in compensatory placental growth and the maintenance of fetal normoxemia (PaO2>17 mmHg). Based on fetal PaO₂, Cx, and Control ewes were assigned to either a normoxemic fetal group (Nx) or a hypoxemic fetal group (Hx) in late gestation, resulting in 4 groups. Cx resulted in a decrease in the volumes of fetal and maternal connective tissues in the placenta and increased placental mRNA expression of IGF2, vascular endothelial growth factor (VEGF), VEGFR-2, ANGPT2, and TIE2 There were reduced volumes of trophoblast, maternal epithelium, and maternal connective tissues in the placenta and a decrease in placental GLUT1 and 11βHSD2 mRNA expression in the Hx compared to Nx groups. Our data show that early restriction of placental growth has effects on morphological and functional characteristics of the placenta in late gestation, independent of whether the fetus becomes hypoxemic. Similarly, there is a distinct set of placental changes that are only present in fetuses that were hypoxemic in late gestation, independent of whether Cx occurred. Thus, we provide further understanding of the different placental cellular and molecular mechanisms that are present in early placental restriction and in the emergence of later placental insufficiency.Song Zhang, Paige Barker, Kimberley J. Botting, Claire T. Roberts, Christine M. McMillan, Isabella Caroline McMillen, Janna L. Morriso

Maternal Obesity and the Early Origins of Childhood Obesity: Weighing Up the Benefits and Costs of Maternal Weight Loss in the Periconceptional Period for the Offspring

There is a need to understand the separate or interdependent contributions of maternal prepregnancy BMI, gestational weight gain, glycaemic control, and macronutrient intake on the metabolic outcomes for the offspring. Experimental studies highlight that there may be separate influences of maternal obesity during the periconceptional period and late gestation on the adiposity of the offspring. While a period of dietary restriction in obese mothers may ablate the programming of obesity, it is associated with an activation of the stress axis in the offspring. Thus, maternal obesity may result in epigenetic changes which predict the need for efficient fat storage in postnatal life, while maternal weight loss may lead to epigenetic changes which predict later adversity. Thus, development of dietary interventions for obese mothers during the periconceptional period requires a greater evidence base which allows the effective weighing up of the metabolic benefits and costs for the offspring

Regulation of microRNA during cardiomyocyte maturation in sheep.

BACKGROUND: There is a limited capacity to repair damage in the mammalian heart after birth, which is primarily due to the inability of cardiomyocytes to proliferate after birth. This is in contrast to zebrafish and salamander, in which cardiomyocytes retain the ability to proliferate throughout life and can regenerate their heart after significant damage. Recent studies in zebrafish and rodents implicate microRNA (miRNA) in the regulation of genes responsible for cardiac cell cycle progression and regeneration, in particular, miR-133a, the miR-15 family, miR-199a and miR-590. However, the significance of these miRNA and miRNA in general in the regulation of cardiomyocyte proliferation in large mammals, including humans, where the timing of heart development relative to birth is very different than in rodents, is unclear. To determine the involvement of miRNA in the down-regulation of cardiomyocyte proliferation occurring before birth in large mammals, we investigated miRNA and target gene expression in sheep hearts before and after birth. The experimental approach included targeted transcriptional profiling of miRNA and target mRNA previously identified in rodent studies as well as genome-wide miRNA profiling using microarrays. RESULTS: The cardiac expression of miR-133a increased and its target gene IGF1R decreased with increasing age, reaching their respective maximum and minimum abundance when the majority of ovine cardiomyocytes were quiescent. The expression of the miR-15 family members was variable with age, however, four of their target genes decreased with age. These latter profiles are inconsistent with the direct involvement of this family of miRNA in cardiomyocyte quiescence in late gestation sheep. The expression patterns of 'pro-proliferative' miR-199a and miR-590 were also inconsistent with their involvement in cardiomyocyte quiescence. Consequently, miRNA microarray analysis was undertaken, which identified six discrete clusters of miRNA with characteristic developmental profiles. The functions of predicted target genes for the miRNA in four of the six clusters were enriched for aspects of cell division and regulation of cell proliferation suggesting a potential role of these miRNA in regulating cardiomyocyte proliferation. CONCLUSION: The results of this study show that the expression of miR-133a and one of its target genes is consistent with it being involved in the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep. The expression patterns of the miR-15 family, miR-199a and miR-590 were inconsistent with direct involvement in the regulation cardiomyocyte proliferation in sheep, despite studies in rodents demonstrating that their manipulation can influence the degree of cardiomyocyte proliferation. miRNA microarray analysis suggests a coordinated and potentially more complex role of multiple miRNA in the regulation of cardiomyocyte quiescence and highlights significant differences between species that may reflect their substantial differences in the timing of this developmental process

Chronic hypoxemia in late gestation decreases cardiomyocyte number but does not change expression of hypoxia-responsive genes

BACKGROUND: Placental insufficiency is the leading cause of intrauterine growth restriction in the developed world and results in chronic hypoxemia in the fetus. Oxygen is essential for fetal heart development, but a hypoxemic environment in utero can permanently alter development of cardiomyocytes. The present study aimed to investigate the effect of placental restriction and chronic hypoxemia on total number of cardiomyocytes, cardiomyocyte apoptosis, total length of coronary capillaries, and expression of genes regulated by hypoxia. METHODS AND RESULTS: We induced experimental placental restriction from conception, which resulted in fetal growth restriction and chronic hypoxemia. Fetal hearts in the placental restriction group had fewer cardiomyocytes, but interestingly, there was no difference in the percentage of apoptotic cardiomyocytes; the abundance of the transcription factor that mediates hypoxia-induced apoptosis, p53; or expression of apoptotic genes Bax and Bcl2. Likewise, there was no difference in the abundance of autophagy regulator beclin 1 or expression of autophagic genes BECN1, BNIP3, LAMP1, and MAP1LC3B. Furthermore, fetuses exposed to normoxemia (control) or chronic hypoxemia (placental restriction) had similar mRNA expression of a suite of hypoxia-inducible factor target genes, which are essential for angiogenesis (VEGF, Flt1, Ang1, Ang2, and Tie2), vasodilation (iNOS and Adm), and glycolysis (GLUT1 and GLUT3). In addition, there was no change in the expression of PKC-ε, a cardioprotective gene with transcription regulated by hypoxia in a manner independent of hypoxia-inducible factors. There was an increased capillary length density but no difference in the total length of capillaries in the hearts of the chronically hypoxemic fetuses. CONCLUSION: The lack of upregulation of hypoxia target genes in response to chronic hypoxemia in the fetal heart in late gestation may be due to a decrease in the number of cardiomyocytes (decreased oxygen demand) and the maintenance of the total length of capillaries. Consequently, these adaptive responses in the fetal heart may maintain a normal oxygen tension within the cardiomyocyte of the chronically hypoxemic fetus in late gestation.Kimberley J. Botting, I. Caroline McMillen, Heather Forbes, Jens R. Nyengaard, Janna L. Morriso

Impact of maternal overnutrition on gluconeogenic factors and methylation of the phosphoenolpyruvate carboxykinase promoter in the fetal and postnatal liver

Advance online publication 22 January 2014BACKGROUND: Exposure to maternal obesity or hyperglycemia increases the risk of obesity and poor glucose tolerance in the offspring. We hypothesized that maternal overnutrition in late pregnancy would result in (i) lower methylation in the promoter region of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C; PCK1) and (ii) higher expression of hepatic gluconeogenic factors in the fetal and postnatal lamb. METHODS: Ewes were fed 100% (n = 18) or ~155% (n = 17) of energy requirements from 115 d gestation, and livers were collected at ~140 d gestation or 30 d postnatal age. RESULTS: Maternal overnutrition resulted in a decrease in hepatic expression of the mitochondrial form of PEPCK (PEPCK-M; PCK2) but not of PEPCK-C or glucose-6-phosphatase (G6PHOS) before and after birth. Hepatic expression of peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1), peroxisome proliferator-activated receptor α (PPARα), PEPCK-C, G6PHOS, and 11β hydroxysteroid dehydrogenase type 1 (11βHSD1), but not PEPCK-M, was higher in the postnatal lamb compared with that in the fetal lamb. The level of PCK1 methylation was paradoxically approximately twofold higher in the postnatal liver compared with that in the fetal liver. CONCLUSION: Maternal overnutrition programs a decrease in hepatic PEPCK-M in the offspring and as ~50% of total hepatic PEPCK is PEPCK-M, the longer-term consequences of this decrease may be significant.Leewen Rattanatray, Beverly S. Muhlhausler, Lisa M. Nicholas, Janna L. Morrison and I. Caroline McMille

The effect of placental restriction on insulin signaling and lipogenic pathways in omental adipose tissue in the postnatal lamb

Intrauterine growth restriction (IUGR) followed by accelerated growth after birth is associated with an increased risk of abdominal (visceral) obesity and insulin resistance in adult life. The aim of the present study was to determine the impact of IUGR on mRNA expression and protein abundance of insulin signaling molecules in one of the major visceral fat depots, the omental adipose depot. IUGR was induced by placental restriction, and samples of omental adipose tissue were collected from IUGR (n = 9, 5 males, 4 females) and Control (n = 14, 8 males, 6 females) neonatal lambs at 21 days of age. The mRNA expression of the insulin signaling molecules, AMP-kinase (AMPK) and adipogenic/lipogenic genes was determined by qRT-PCR, and protein abundance by Western Blotting. AMPKα2 mRNA expression was increased in male IUGR lambs (0.015 ± 0.002 v. 0.0075 ± 0.0009, P < 0.001). The proportion of the AMPK pool that was phosphorylated (%P-AMPK) was lower in IUGR lambs compared with Controls independent of sex (39 ± 9% v. 100 ± 18%, P < 0.001). The mRNA expression and protein abundance of insulin signaling proteins and adipogenic/lipogenic genes was not different between groups. Thus, IUGR is associated with sex-specific alterations in the mRNA expression of AMPKα2 and a reduction in the percentage of the total AMPK pool that is phosphorylated in the omental adipose tissue of neonatal lambs, before the onset of visceral obesity. These molecular changes would be expected to promote lipid accumulation in the omental adipose depot and may therefore contribute to the onset of visceral adiposity in IUGR animals later in life.S. Lie, J. A. Duffield, I. C. McMillen, J. L. Morrison, S. E. Ozanne, C. Pilgrim and B. S. Muhlhausle

Placental adaptations in growth restriction

The placenta is the primary interface between the fetus and mother and plays an important role in maintaining fetal development and growth by facilitating the transfer of substrates and participating in modulating the maternal immune response to prevent immunological rejection of the conceptus. The major substrates required for fetal growth include oxygen, glucose, amino acids and fatty acids, and their transport processes depend on morphological characteristics of the placenta, such as placental size, morphology, blood flow and vascularity. Other factors including insulin-like growth factors, apoptosis, autophagy and glucocorticoid exposure also affect placental growth and substrate transport capacity. Intrauterine growth restriction (IUGR) is often a consequence of insufficiency, and is associated with a high incidence of perinatal morbidity and mortality, as well as increased risk of cardiovascular and metabolic diseases in later life. Several different experimental methods have been used to induce placental insufficiency and IUGR in animal models and a range of factors that regulate placental growth and substrate transport capacity have been demonstrated. While no model system completely recapitulates human IUGR, these animal models allow us to carefully dissect cellular and molecular mechanisms to improve our understanding and facilitate development of therapeutic interventions.Song Zhang, Timothy R.H. Regnault, Paige L. Barker, Kimberley J. Botting, Isabella C. McMillen, Christine M. McMillan, Claire T. Roberts and Janna L. Morriso