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.

The transition from fetal growth restriction to accelerated postnatal growth: a potential role for insulin signalling in skeletal muscle.

A world-wide series of epidemiological and experimental studies have demonstrated that there is an association between being small at birth, accelerated growth in early postnatal life and the emergence of insulin resistance in adult life. The aim of this study was to investigate why accelerated growth occurs in postnatal life after in utero growth restriction. Samples of quadriceps muscle were collected at approximately 140 days gestation (term approximately 150 days gestation) from normally grown fetal lambs (Control, n = 7) and from growth restricted fetal lambs (placentally restricted: PR, n = 8) and from Control (n = 14) and PR (n = 9) lambs at 21 days after birth. The abundance of the insulin and IGF1 receptor protein was higher in the quadriceps muscle of the PR fetus, but there was a lower abundance of the insulin signalling molecule PKC, and GLUT4 protein in the PR group. At 21 days of postnatal age, insulin receptor abundance remained higher in the muscle of the PR lamb, and there was also an up-regulation of the insulin signalling molecules, PI3Kinase p85, Akt1 and Akt2 and of the GLUT4 protein in the PR group. Fetal growth restriction therefore results in an increased abundance of the insulin receptor in skeletal muscle, which persists after birth when it is associated with an upregulation of insulin signalling molecules and the glucose transporter, GLUT4. These data provide evidence that the origins of the accelerated growth experienced by the small baby after birth lie in the adaptive response of the growth restricted fetus to its low placental substrate supply.

Maternal overnutrition suppresses the phosphorylation of 5'-AMP-activated protein kinase in liver, but not skeletal muscle, in the fetal and neonatal sheep.

Epidemiological studies have shown that infants exposed to an increased supply of nutrients before birth are at increased risk of type 2 diabetes in later life. We have investigated the hypothesis that fetal overnutrition results in reduced expression and phosphorylation of the cellular fuel sensor, AMP-activated kinase (AMPK) in liver and skeletal muscle before and after birth. From 115 days gestation, ewes were fed either at or approximately 55% above maintenance energy requirements. Postmortem was performed on lamb fetuses at 139-141 days gestation (n = 14) and lambs at 30 days of postnatal age (n = 21), and liver and quadriceps muscle were collected at each time point. The expression of AMPKalpha1 and AMPKalpha2 mRNA was determined by quantitative RT-PCR (qRT-PCR). The abundance of AMPKalpha and phospho-AMPKalpha (P-AMPKalpha) was determined by Western blot analysis, and the proportion of the total AMPKalpha pool that was phosphorylated in each sample (%P-AMPKalpha) was determined. The ratio of AMPKalpha2 to AMPKalpha1 mRNA expression was lower in fetuses compared with lambs in both liver and muscle, independent of maternal nutrition. Hepatic %P-AMPKalpha was lower in both fetuses and lambs in the Overfed group and %P-AMPKalpha in the lamb liver was inversely related to plasma glucose concentrations in the first 24 h after birth (r = 0.73, P < 0.025). There was no effect of maternal overnutrition on total AMPKalpha or P-AMPKalpha abundance in liver or skeletal muscle. We have, therefore, demonstrated that AMPKalpha responds to signals of increased nutrient availability in the fetal liver. Suppression of hepatic AMPK phosphorylation may contribute to increased glucose production, and basal hyperglycemia, present in lambs of overfed ewes in early postnatal life.

Birth weight and gender determine expression of adipogenic, lipogenic and adipokine genes in perirenal adipose tissue in the young adult sheep.

Epidemiological studies have demonstrated that low birth weight is associated with an increased incidence of visceral obesity and metabolic disorders in later life. In the present study, we have determined the impact of birth weight and gender on gene expression in visceral adipose tissue (VAT) in the young adult sheep. Lambs (n=19, birth weight range 2.6-7.55 kg) were born at term and growth monitored for 22.4+/-0.2 weeks, when body composition was determined by Dual X-ray Absorptiometry (DXA) and samples of VAT and subcutaneous (SCAT) adipose tissue collected. Plasma samples were collected at post-mortem for the determination of free fatty acids (FFA), glucose and insulin concentrations. Peroxisome-Proliferator Activated Receptor-gamma (PPARgamma), glycerol-3-phosphate dehydrogenase (G3PDH), lipoprotein lipase (LPL), adiponectin and leptin mRNA expression was determined by qRT-PCR. Fractional growth rate in postnatal weeks 1-3 was inversely related to birth weight in both males and females (R2=0.22, P<0.05, n=19). PPARgamma mRNA expression in VAT, but not SCAT, was inversely related to birth weight (R2=0.60, P<0.01, n=18). In males, but not females, PPARgamma mRNA in VAT was directly related to G3PDH mRNA expression (R2=0.69, P<0.01, n=9). Plasma FFA concentrations were inversely related to birth weight in both males and females (R2=0.22, P<0.05, n=19). These findings demonstrate that low birth weight is associated with an increased expression of a key adipogenic factor in visceral adipose tissue in young adulthood. In males, this is associated with an increased expression of lipogenic genes, and this may contribute to the increased propensity for visceral obesity in low birth weight males compared to females.

Increased maternal nutrition increases leptin expression in perirenal and subcutaneous adipose tissue in the postnatal lamb.

The present study tested the hypothesis that exposure to an increased level of maternal nutrition before birth results in altered expression of adipogenic, lipogenic, and adipokine genes in adipose tissue in early postnatal life. Pregnant ewes were fed either at or approximately 50% above maintenance energy requirements during late pregnancy, and quantitative RT-PCR was used to measure peroxisome proliferator-activated receptor (PPAR)-gamma, lipoprotein lipase (LPL), glycerol-3-phosphate-dehydrogenase (G3PDH), adiponectin, and leptin mRNA expression in perirenal (PAT) and sc adipose tissue (SCAT) in the offspring on postnatal d 30. Relative SCAT mass was higher in lambs of well-fed ewes (40.0 +/- 4.0 vs. 22.8 +/- 3.3 g/kg, P < 0.05) and was directly related to plasma insulin in the first 24 h after birth and to G3PDH and LPL expression. The expression of leptin mRNA in both the SCAT and PAT depots was higher (P < 0.05) in lambs of well-fed ewes. PPARgamma adiponectin, LPL, and G3PDH mRNA expression were not, however, different between well-fed and control groups in either depot. Relative PPARgamma expression in SCAT was directly related to plasma insulin concentrations in the first 24 h after birth (r(2) = 0.23; P < 0.05), and G3PDH and LPL expressions were also positively correlated with PPARgamma expression (r(2) = 0.27; P < 0.05). We have demonstrated that exposure to increased prenatal nutrition increases leptin expression at 1 month of age in both PAT and SCAT. The results of this study provide evidence that the nutritional environment before and immediately after birth can influence the development of adipose tissue in early postnatal life.

Increased maternal nutrition stimulates peroxisome proliferator activated receptor-gamma, adiponectin, and leptin messenger ribonucleic acid expression in adipose tissue before birth.

During fetal life, adipose tissue is predominantly comprised of brown or thermogenic adipocytes and there is a transition to white, lipid-storing adipocytes after birth concomitant with the onset of suckling. In pregnancies complicated by gestational diabetes, the fetus is hyperglycemic, has an increased fat mass, and is at increased risk of obesity in later life. In the present study, we have investigated the hypothesis that exposure to increased maternal nutrition during late gestation results in increased expression of genes that regulate adipogenesis and lipogenesis in perirenal fat in fetal sheep. Pregnant ewes were fed either at or approximately 55% above maintenance energy requirements during late pregnancy and quantitative RT-PCR was used to measure peroxisome proliferator-activated receptor gamma, lipoprotein lipase, glycerol-3-phosphate dehydrogenase, adiponectin, and leptin mRNA expression. We report that exposure to metabolic and hormonal signals of increased nutrition before birth results in an increase in the expression of the adipogenic factor, peroxisome proliferator-activated receptor gamma, and in lipoprotein lipase, adiponectin, and leptin mRNA expression in fetal perirenal fat. We propose that an increase in maternal, and hence fetal, nutrition results in a precocial increase in adipogenic, lipogenic, and adipokine gene expression in adipose tissue and that these changes may be important in the development of obesity in later life.

Increased maternal nutrition alters development of the appetite-regulating network in the brain.

Individuals exposed to an increased nutrient supply before birth have a high risk of becoming obese children and adults. It has been proposed that exposure of the fetus to high maternal nutrient intake results in permanent changes within the central appetite regulatory network. No studies, however, have investigated the impact of increased maternal nutrition on the appetite regulatory network in species in which this network develops before birth, as in the human. In the present study, pregnant ewes were fed a diet which provided 100% (control, n = 8) or approximately 160% (well-fed, n = 8) of metabolizable energy requirements. Ewes were allowed to lamb spontaneously, and lambs were sacrificed at 30 days of postnatal age. All fat depots were dissected and weighed, and expression of the appetite-regulating neuropeptides and the leptin receptor (OBRb) were determined by in situ hybridization. Lambs of well-fed ewes had higher glucose (Glc) concentrations during early postnatal life (F = 5.93, P<0.01) and a higher relative subcutaneous (s.c.) fat mass at 30 days of age (34.9+/-4.7 g/kg vs. 22.8+/-3.3 g/kg; P<0.05). The hypothalamic expression of pro-opiomelanocortin was higher in lambs of well-fed ewes (0.48+/-0.09 vs. 0.28+/-0.04, P<0.05). In lambs of overnourished mothers, but not in controls, the expression of OBRb was inversely related to total relative fat mass (r2 = 0.50, P = 0.05, n = 8), and the direct relationship between the expression of the central appetite inhibitor CART and fat mass was lost. The expression of neuropeptide Y and AGRP was inversely related to total relative fat mass (NPY, r2 = 0.28, P<0.05; agouti-related peptide, r2 = 0.39, P<0.01). These findings suggest that exposure to increased nutrition before birth alters the responses of the central appetite regulatory system to signals of increased adiposity after birth.

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.