Jaime A. Duffield

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 ∼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 proopiomelanocortin was higher in lambs of well‐fed ewes (0.48±0.09 vs. 0.28±0.04, P<0.05). In lambs of over‐nourished 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.—Muhlhausler, B. S., Adam, C. L., Findlay, P. A., Duffield, J. A., and McMillen, I. C. Increased maternal nutrition alters development of the appetite‐regulating network in the brain. FASEB J. 20, E556–E565 (2006)

Fetal growth restriction, catch-up growth and the early origins of insulin resistance and visceral obesity.

There is an association between growing slowly before birth, accelerated growth in early postnatal life and the emergence of insulin resistance, visceral obesity and glucose intolerance in adult life. In this review we consider the pathway through which intrauterine growth restriction (IUGR) leads to the initial increase in insulin sensitivity and to catch-up growth. We also discuss the importance of the early insulin environment in determining later visceral adiposity and the intrahepatic mechanisms that may result in the emergence of glucose intolerance in a subset of IUGR infants. We present evidence that a key fetal adaptation to poor fetal nutrition is an upregulation of the abundance of the insulin receptor in the absence of an upregulation of insulin signalling in fetal skeletal muscle. After birth, however, there is an upregulation in the abundance of the insulin receptor and the insulin signalling pathway in the IUGR offspring. Thus, the origins of the accelerated postnatal growth rate experienced by IUGR infants lie in the fetal adaptations to a poor nutrient supply. We also discuss how the intracellular availability of free fatty acids and glucose within the visceral adipocyte and hepatocyte in fetal and neonatal life are critical in determining the subsequent metabolic phenotype of the IUGR offspring. It is clear that a better understanding of the relative contributions of the fetal and neonatal nutrient environment to the regulation of key insulin signalling pathways in muscle, visceral adipose tissue and the liver is required to support the development of evidence-based intervention strategies and better outcomes for the IUGR infant.

The early origins of later obesity: pathways and mechanisms.

Excess bodyweight is the sixth most important risk factor contributing to the overall burden of disease worldwide. In excess of a billion adults and 10% of all children are now classified as overweight or obese. The main adverse consequences of obesity are the metabolic syndrome, cardiovascular disease and type 2 diabetes and a diminished average life expectancy. It has been argued that the complex pathological processes underlying obesity reflect environmental and genetic interactions, and individuals from disadvantaged communities seem to have greater risks than more affluent individuals partly because of fetal and postnatal programming interactions. Abundant evidence indicates that the obesity epidemic reflects progressive secular and age-related decreases in physical activity, together with passive over-consumption of energy dense foods despite neurobiological processes designed to regulate energy balance. The difficulty in treating obesity, however, highlights the deficits in our current understanding of the pathophysiology which underlies the initiation and chronic nature of this disorder. Large population based studies in Europe and North America in healthy women and in women with gestational diabetes have demonstrated that there are clear relationships between maternal and fetal nutrient supply, fetal growth patterns and the subsequent risk of obesity and glucose intolerance in childhood and adult life. In this review we discuss the impact of fetal nutrition on the biology of the developing adipocyte and brain and the growing evidence base supporting an intergenerational cycle of obesity.

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 ∼140 days gestation (term ∼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 PKCζ, 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.

Fetal growth restriction and the programming of heart growth and cardiac insulin-like growth factor 2 expression in the lamb.

Non‐Technical Summary  Cardiovascular disease is responsible for 30% of deaths worldwide and epidemiological data demonstrate that poor growth before birth is associated with an increased risk of heart disease in adult life. We show that in response to reduced placental substrate supply there is an increase in cardiac insulin‐like growth factor‐2 (IGF‐2) and the IGF‐2 receptor (IGF‐2R) in the fetus. Importantly, this effect is programmed because it is also present after birth in the lamb at 21 days of age. We also show that the increase in IGF‐2 and IGF‐2R gene expression is not epigenetically regulated through the IGF‐2/H19 or IGF‐2R methylation process. This study places the IGF‐2 receptor signalling pathway as a prime candidate for mediating cardiac hypertrophy in fetal growth restriction before and after birth.

Placental restriction of fetal growth decreases IGF1 and leptin mRNA expression in the perirenal adipose tissue of late gestation fetal sheep

Placental restriction (PR) of fetal growth results in a low birth weight and an increased visceral fat mass in postnatal life. We investigated whether PR alters expression of genes that regulate adipogenesis [IGF1, IGF1 receptor (IGF1R), IGF2, IGF2R, proliferator-activated receptor-gamma, retinoid-X-receptor-alpha], adipocyte metabolism (lipoprotein lipase, G3PDH, GAPDH) and adipokine signaling (leptin, adiponectin) in visceral adipose tissue before birth. PR was induced by removal of the majority of endometrial caruncles in nonpregnant ewes before mating. Fetal blood samples were collected from 116 days gestation, and perirenal visceral adipose tissue (PAT) was collected from PR and control fetuses at 145 days. PAT gene expression was measured by quantitative RT-PCR. PR fetuses had a lower weight (PR 2.90 +/- 0.32 kg; control, 5.12 +/- 0.24 kg; P < 0.0001), mean gestational arterial Po(2) (P < 0.0001), plasma glucose (P < 0.01), and insulin concentrations (P < 0.02), than controls. The expression of IGF1 mRNA in PAT was lower in the PR fetuses (PR, 0.332 +/- 0.063; control, 0.741 +/- 0.083; P < 0.01). Leptin mRNA expression in PAT was also lower in PR fetuses (PR, 0.077 +/- 0.009; control, 0.115 +/- 0.013; P < 0.05), although there was no difference in the expression of other adipokine or adipogenic genes in PAT between PR and control fetuses. Thus, restriction of placental and hence, fetal substrate supply results in decreased IGF1 and leptin expression in fetal visceral adipose tissue, which may alter the functional development of the perirenal fat depot and contribute to altered leptin signaling in the growth-restricted newborn and the subsequent emergence of an increased visceral adiposity.

Intrauterine Growth Restriction and the Sex Specific Programming of Leptin and Peroxisome Proliferator-Activated Receptor γ (PPARγ) mRNA Expression in Visceral Fat in the Lamb

Being born small is associated with an increased risk of visceral obesity and insulin resistance in adult life. We have investigated the effect of IUGR on adipogenic and lipogenic gene expression in visceral fat in the lamb at 3 wk of age. Perirenal fat mass, but not adipocyte size was greater in females than males, independent of birth weight. Plasma insulin concentrations during the first 24 h after birth predicted the size of the adipocytes and expression of adiponectin in visceral adipose tissue in both males and females. In females, plasma nonesterified fatty acids (NEFA) concentrations during the first 24 h after birth were directly related to peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression in the perirenal fat depot at 3 wk of age. In the males, in contrast to the females, PPARγ and leptin expression in perirenal visceral fat were significantly lower in IUGR compared with control lambs. Thus, the early nutritional environment programs adipocyte growth and gene expression in visceral adipose tissue. The differential effect of sex and IUGR on PPARγ and leptin expression in visceral fat may be important in the subsequent development of visceral obesity and the insulin resistant phenotype in later 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.

Alteration of cardiac glucose metabolism in association to low birth weight: Experimental evidence in lambs with left ventricular hypertrophy

OBJECTIVE Intrauterine growth restriction that results in low birth weight (LBW) has been linked to the onset of pathological cardiac hypertrophy. An altered transition from a fetal to an adult energy metabolism phenotype, with increased reliance on glucose rather than fatty acids for energy production, could help explain this connection. We have therefore investigated cardiac metabolism in relation to left ventricular hypertrophy in LBW lambs, at 21days after birth. MATERIALS/METHODS The expression of regulatory molecules involved in cardiac glucose and fatty acid metabolism was measured using real-time PCR and Western blotting. A section of the left ventricle was fixed for Periodic Acid Schiff staining to determine tissue glycogen content. RESULTS There was increased abundance of insulin signalling pathway proteins (phospho-insulin receptor, insulin receptor and phospho-Akt) and the glucose transporter (GLUT)-1, but no change in GLUT-4 or glycogen content in the heart of LBW compared to ABW lambs. There was, however, increased abundance of cardiac pyruvate dehydrogenase kinase 4 (PDK-4) in LBW compared to ABW lambs. There were no significant changes in the mRNA expression of components of the peroxisome proliferator activated receptor regulatory complex or proteins involved in fatty acid metabolism. CONCLUSION We concluded that LBW induced left ventricular hypertrophy was associated with increased GLUT-1 and PDK-4, suggesting increased glucose uptake, but decreased efficacy for the conversion of glucose to ATP. A reduced capacity for energy conversion could have significant implications for vulnerability to cardiovascular disease in adults who are born LBW.

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.