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Type: Thesis
Title: Role of microRNA in early life placental programming of insulin resistance and metabolic health.
Author: Harryanto, Himawan
Issue Date: 2014
School/Discipline: School of Paediatrics and Reproductive Health
Abstract: Intrauterine growth restriction (IUGR) is associated with insulin resistance and diabetes, particularly later in adult life. Placental restriction is a common cause of IUGR, this condition induces insulin resistance and/ or insulin deficiency and consequently, impaired glucose tolerance in offspring, in the sheep and rat. Reduced expression of insulin signalling genes and that of their key metabolic targets in insulin sensitive tissues and of some molecular determinants of pancreatic β-cell insulin secretion and mass, contributes to impaired insulin action in offspring, in experimental and human IUGR. However, the underlying molecular mechanisms whereby IUGR alters the molecular profile of insulin sensitive and secreting tissues in later life are largely unknown. The studies described in this thesis examine the potential role of microRNAs (miRNAs) in the developmental programming of impaired insulin action in IUGR offspring. MiRNAs are short single-stranded RNAs (22 nucleotide in length), which are able to reduce the translation and/or abundance of mRNA and protein of targets. Each miRNA is predicted to regulate the abundance of many targets in co-ordinated networks to modify function, providing a potentially powerful pathway for developmental programming to influence later phenotype. Here, IUGR was induced by restricting placental growth and development surgically in sheep (pre-conception removal of most implantation sites) or in the rat (ligation of uterine blood vessels in late gestation). In each species, the effect of placental restriction and IUGR on miRNA expression and expression of key predicted targets, including that of insulin signalling and key metabolic genes in the insulin sensitive tissues: liver, skeletal muscle and adipose (perirenal in sheep and omental in rat), in adult offspring were characterised. The effect of placental restriction on pancreatic miRNA expression in adult offspring was also determined in the sheep. Placental restriction and IUGR mostly increased miRNA expression in insulin sensitive tissues of the adult sheep and in a sex specific manner, suggesting the potential for increased repression of the translation or abundance of their molecular targets and related functions. The liver, followed by skeletal muscle and adipose tissue, showed the greatest susceptibility in terms of numbers of miRNAs with altered expression following placental restriction. In males, placental restriction increased hepatic expression of eight miRNAs by ~1.5-3.5-fold, with differential expression of four independently confirmed by qPCR (hsa-miR-1, hsa-miR-21, hsa-miR-142-3p and hsa-miR-144). Each of these four miRNAs was predicted to target molecules involved in insulin signalling, metabolism and hepatic disease. The latter included p85α, Pparα, Igf1, Foxo3 and Acox1, all exhibiting reduced hepatic expression (~2.3-4.0 fold) following placental restriction in males, with the abundance of hsa-miR-1, hsa-miR-142-3p and hsa-miR-144 correlating negatively with Acox1 expression. Thus, placental restriction co-ordinately alters hepatic expression of miRNAs and predicted targets related to non-alcoholic fatty liver disease (NAFLD) in adult male offspring in sheep. Reduced hepatic expression of Pparα (regulates lipid catabolism), and Acox1 (peroxisomal fatty acid β-oxidation) is characterised to promote the development of NAFLD, increasingly common following fetal growth restriction in humans, and miRNAs may partly mediate this prenatal programming of NAFLD. In the sheep, placental restriction increased vastus lateralis expression of seven miRNAs by ~1.23-2.04 fold, with differential expression of two independently confirmed by qPCR (hsa-miR-17-5p and hsa-miR-376b). Both of these two miRNAs were predicted to regulate Pparα expression, which tended to negative correlate with that of hsa-miR-376b (r = -0.617, P-value: 0.052) and hsa-miR-17-5p (r = -0.533, P-value: 0.087), in placentally restricted female offspring. Placental restriction also increased perirenal fat hsa-miR-451 expression in female offspring. This miRNA is predicted to regulate a network that is involved in lipid metabolism, molecular transport and small molecule biochemistry in adipose tissue. Furthermore, perirenal fat expression of hsa-miR-451 was correlated positively with in vivo insulin sensitivity of free fatty acids in control offspring (r = 0.687, P = 0.020, n = 11), but not in placentally restricted offspring. In the rat, placental restriction impaired insulin secretion in adult offspring, while insulin sensitivity was enhanced in young adult offspring, which then disappeared with aging, particularly in females. Placental restriction and IUGR also mostly increased miRNA expression in the insulin sensitive tissues of older adult offspring and usually in a sex specific manner, with omental fat the most affected, followed by skeletal muscle and liver. Placental restriction and IUGR reduced hepatic expression of the insulin signalling molecule, p110β in female offspring and that of related molecules, Slc2a2 and Igf1, in male offspring. Placental restriction also increased hepatic rno-miR-126 expression, in female offspring and is predicted to target molecules involved in lipid metabolism, molecular transport and small molecule biochemistry. Placental restriction and IUGR reduced omental fat expression of Irs1, Irs2 and Slc2a4 in male offspring and increased that of rno-miR-18a, rno-miR-142-3p, rno-miR-19b, rno-miR-21, rno-miR-20b and mmu-miR-106a. The latter are predicted to target insulin signalling but also small molecule biochemistry, lipid metabolism and its regulation, including similar pathways to those targeted by IUGR altered miRNAs in liver of adult offspring in sheep. Expression of omental fat rno-miR-18a was found to be negatively correlated with Slc2a4 in placentally restricted offspring overall (r = -0.451, P = 0.040). Placental restriction also alters the pancreatic expression of three miRNAs in adult sheep offspring, hsa-miR-339-5p in males, rno-miR-331* in females and hsa-miR-513a-3p in both sexes. Furthermore, the predicted molecular and functional targets of these differentially expressed miRNAs and predicted functional outcomes following placental restriction mirror the previously reported sex differences in β-cell insulin secretory function and mass in the placentally restricted adult sheep. We found that hsa-miR-339-5p was predicted to regulate PLEKHH1 and PAK6, proteins which are essential to maintain the development of the pancreas and/or differentiation of islets. Down-regulation of both rno-miR-331* and hsa-miR-513a-3p in the pancreas of placentally restricted female offspring would be expected to indirectly up-regulate Pdx1 expression, and potentially contribute to the increased number of β-cells per islet they exhibit. Overall, placental restriction and IUGR mostly increase abundance of miRNAs in key insulin sensitive tissues of adult offspring in both sheep and the rat, with sex-specific and tissue-specific differences. Nevertheless, the networks targeted by miRNAs differentially expressed following IUGR in such tissues, share common functions and pathways, both across tissues and species, including small molecule biochemistry, lipid metabolism, carbohydrate metabolism and molecular transport. Of interest, regardless of the tissues and species, placental restriction generally increased expression of miR-142-3p, miR-1, miR-21 and miR-17-5p. Of note, expression of hsa-miR-1 and hsa-miR-21 were each up-regulated in both liver and vastus lateralis in sheep offspring. These up-regulations of common miRNAs expression could be due to alteration of epigenetic mechanism affected by placental restriction, such as DNA methylation, or common systemic regulations of their expression that has been ‘programmed’ due to placental restriction. Therefore, placental restriction of fetal growth does alter expression of miRNAs and their networks involving insulin signalling and metabolism in key insulin sensitive tissues in the adult. The mechanism underlying this and the extent to which they contribute to overall developmental programming of metabolic dysfunction warrant for future investigation.
Advisor: Owens, Julie Anne
De Blasio, Miles Jonathon
Grant, P.
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2014
Keywords: IUGR; placental restriction; microRNA; insulin resistance
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