Anne-Maj Samuelsson

Diet-Induced Obesity in Female Mice Leads to Offspring Hyperphagia, Adiposity, Hypertension, and Insulin Resistance A Novel Murine Model of Developmental Programming

Maternal obesity is increasingly prevalent and may affect the long-term health of the child. We investigated the effects of maternal diet-induced obesity in mice on offspring metabolic and cardiovascular function. Female C57BL/6J mice were fed either a standard chow (3% fat, 7% sugar) or a palatable obesogenic diet (16% fat, 33% sugar) for 6 weeks before mating and throughout pregnancy and lactation. Offspring of control (OC) and obese dams (OO) were weaned onto standard chow and studied at 3 and 6 months of age. OO were hyperphagic from 4 to 6 weeks of age compared with OC and at 3 months locomotor activity was reduced and adiposity increased (abdominal fat pad mass; P<0.01). OO were heavier than OC at 6 months (body weight, P<0.05). OO abdominal obesity was associated with adipocyte hypertrophy and altered mRNA expression of &bgr;-adrenoceptor 2 and 3, 11&bgr;HSD-1, and PPAR-&ggr; 2. OO showed resistance artery endothelial dysfunction at 3 months, and were hypertensive, as assessed by radiotelemetry (nighttime systolic blood pressure at 6 months [mm Hg] mean±SEM, male OO, 134±1 versus OC, 124±2, n=8, P<0.05; female OO, 137±2 versus OC, 122±4, n=8, P<0.01). OO skeletal muscle mass (tibialis anterior) was significantly reduced (P<0.01) OO fasting insulin was raised at 3 months and by 6 months fasting plasma glucose was elevated. Exposure to the influences of maternal obesity in the developing mouse led to adult offspring adiposity and cardiovascular and metabolic dysfunction. Developmentally programmed hyperphagia, physical inactivity, and altered adipocyte metabolism may play a mechanistic role.

Maternal Obesity Induced by Diet in Rats Permanently Influences Central Processes Regulating Food Intake in Offspring

Hypothalamic systems which regulate appetite may be permanently modified during early development. We have previously reported hyperphagia and increased adiposity in the adult offspring of rodents fed an obesogenic diet prior to and throughout pregnancy and lactation. We now report that offspring of obese (OffOb) rats display an amplified and prolonged neonatal leptin surge, which is accompanied by elevated leptin mRNA expression in their abdominal white adipose tissue. At postnatal Day 30, before the onset of hyperphagia in these animals, serum leptin is normal, but leptin-induced appetite suppression and phosphorylation of STAT3 in the arcuate nucleus (ARC) are attenuated; the level of AgRP-immunoreactivity in the hypothalamic paraventricular nucleus (PVH), which derives from neurones in the ARC and is developmentally dependent on leptin, is also diminished. We hypothesise that prolonged release of abnormally high levels of leptin by neonatal OffOb rats leads to leptin resistance and permanently affects hypothalamic functions involving the ARC and PVH. Such effects may underlie the developmental programming of hyperphagia and obesity in these rats.

Maternal obesity during pregnancy and lactation programs the development of offspring non-alcoholic fatty liver disease in mice.

BACKGROUND & AIMS Obesity induced, non-alcoholic fatty liver disease (NAFLD), is now the major cause in affluent countries, of the spectrum of steatosis-to-cirrhosis. Obesity and NAFLD rates in reproductive age women, and adolescents, are rising worldwide. Our hypothesis was that maternal obesity and lactation transmit to the offspring a pre-disposition to dysmetabolism, obesity and NAFLD. METHODS Female mice were fed standard or obesogenic chow, before, throughout pregnancy, and during lactation. The critical developmental period was studied by cross-fostering offspring of lean and obese dams. Offspring were then weaned onto standard chow and studied at 3months. Read-outs included markers of metabolic dysfunction, biochemical and histological indicators of NAFLD, induction of liver fibrogenesis, and activation of pro-fibrotic pathways. Mechanisms involved in programming a dysmetabolic and NAFLD phenotype were investigated by assaying breast milk components. RESULTS Offspring of obese dams had a dysmetabolic, insulin resistant and NAFLD phenotype compared to offspring of lean dams. Offspring of lean dams that were suckled by obese dams showed an exaggerated dysmetabolic and NAFLD phenotype, with increased body weight, as well as increased levels of insulin, leptin, aspartate transaminase, interleukin-6, tumour necrosis factor-alpha, liver triglycerides, steatosis, hepatic fibrogenesis, renal norepinephrine, and liver alpha1-D plus beta1-adrenoceptors, indicative of sympathetic nervous system activation. Obese dams also had raised breast milk leptin levels compared to lean dams. CONCLUSIONS Maternal obesity programs development of a dysmetabolic and NAFLD phenotype, which is critically dependent on the early postnatal period and possibly involving alteration of hypothalamic appetite nuclei signalling by maternal breast milk and neonatal adipose tissue derived, leptin.

Interleukin-6 in the Maternal Circulation Reaches the Rat Fetus in Mid-gestation

Maternal systemic infection during pregnancy may expose the fetus to infectious agents and high levels of mediators of the resulting inflammatory response, such as IL-6 (IL-6). Increased fetal and maternal levels of IL-6 have been associated with adverse neonatal outcome but might also stress the fetus and contribute to cardiovascular and neuroendocrine dysfunction in adulthood. It is unclear whether interleukines cross the placental barrier, although this matter has been little studied. The aim of this study was therefore to investigate if IL-6 administered to pregnant rats in vivo is transferred to the fetus. We injected 125I IL-6 i.v. to pregnant dams at gestation day 11–13 (mid-gestation) or 17–19 (late gestation). We found 125I-IL-6 in the exposed fetuses as well as in amniotic fluids. Fetal 125I-IL-6 levels were markedly higher in animals injected in mid-gestation compared with late pregnancy (p < 0.01). This difference was mirrored in a 15-fold higher unidirectional materno-fetal clearance for 125I-IL-6 in mid-gestation (p < 0.01). We conclude that the permeability of the rat placental barrier to IL-6 is much higher in mid-gestation than in late pregnancy. Maternally derived IL-6 may directly induce fetal injury but also stimulate the release of fetal stress hormones resulting in stimuli or insults in neuroendocrine structures and hormonal axes which might lead to disease at adult age.

Evidence for Sympathetic Origins of Hypertension in Juvenile Offspring of Obese Rats

Maternal obesity in rodents is associated with increased adiposity, impaired glucose tolerance, and hypertension in adult offspring. In this study we investigated the influence of maternal obesity in the rat on blood pressure and blood pressure regulatory pathways in juvenile and adult offspring. Obesity was induced before pregnancy in female Sprague-Dawley rats by feeding a highly palatable energy-dense diet. In juvenile animals (30 days of age), before the onset of obesity and hyperleptinemia, basal nighttime mean arterial pressure was significantly raised in the offspring of obese dams (OffOb) relative to offspring of controls (OffCon; mean arterial pressure, males: OffOb, 121.8±0.6 mm Hg versus OffCon, 115.0±0.5 mm Hg, n=6, P<0.01; females: OffOb, 125.4±0.4 mm Hg versus OffCon, 114.4±0.5 mm Hg, n=6, P<0.001), as was the mean arterial pressure response to restraint stress (P<0.01). The pressor response to a leptin challenge was enhanced in OffOb rats (&Dgr;mean arterial pressure: OffOb, 9.7±0.8 mm Hg versus OffCon, 5.3±1.3 mm Hg; n=8; P<0.05). Renal tissue norepinephrine content (P<0.001) and renin expression (P<0.05) were markedly raised. Analysis of heart rate variability revealed an increased low:high frequency ratio in OffOb versus OffCon rats (P<0.05). At 90 days, hypertension in OffOb rats persisted and was abolished by &agr;1- and &bgr;-adrenergic blockade, and cardiovascular responses to phenylephrine or sodium nitroprusside indicated altered baroreceptor function. The exaggerated pressor response to leptin in OffOb rats was maintained. Hypertension in the offspring of obese rats may arise from persistent sympathoexcitatory hyperresponsiveness acquired in early stages of development.

Maternal obesity programs offspring nonalcoholic fatty liver disease by innate immune dysfunction in mice

The global prevalence of obesity‐induced liver disease (nonalcoholic fatty liver disease; NAFLD) is rising. Suggested causes include a role for in utero influences of maternal obesity compounded by the availability of energy‐dense foods throughout postnatal life. Using a physiologically relevant model, we investigated the role of the innate immune system in liver injury induced by maternal obesity followed by a postnatal obesogenic diet. Female C57BL/6J mice were fed a standard or obesogenic diet before and throughout pregnancy and during lactation. Female offspring were weaned onto a standard or obesogenic diet at 3 weeks postpartum. Biochemical and histological indicators of dysmetabolism, NAFLD and fibrosis, analysis of profibrotic pathways, liver innate immune cells, and reactive oxygen species (ROS) were investigated at 3, 6, and 12 months. Female offspring exposed to a postweaning obesogenic diet (OffCon‐OD) demonstrated evidence of liver injury, which was exacerbated by previous exposure to maternal obesity (OffOb‐OD), as demonstrated by raised alanine aminotransferase, hepatic triglycerides, and hepatic expression of interleukin (IL)‐6, tumor necrosis factor alpha, transforming growth factor beta, alpha smooth muscle actin, and collagen (P < 0.01). Histological evidence of hepatosteatosis and a more‐robust NAFLD phenotype with hepatic fibrosis was observed at 12 months in OffOb‐OD. A role for the innate immune system was indicated by increased Kupffer cell numbers with impaired phagocytic function and raised ROS synthesis (P < 0.01), together with reduced natural killer T cells and raised interleukin (IL)‐12 and IL‐18. Conclusion: Maternal obesity in the context of a postnatal hypercalorific obesogenic diet aggressively programs offspring NAFLD associated with innate immune dysfunction, resulting in a comprehensive phenotype that accurately reflects the human disease. (HEPATOLOGY 2013)

Maternal obesity and the developmental programming of hypertension: a role for leptin

Mother–child cohort studies have established that both pre‐pregnancy body mass index (BMI) and gestational weight gain are independently associated with cardio‐metabolic risk factors in young adult offspring, including systolic and diastolic blood pressure. Animal models in sheep and non‐human primates provide further evidence for the influence of maternal obesity on offspring cardiovascular function, whilst recent studies in rodents suggest that perinatal exposure to the metabolic milieu of maternal obesity may permanently change the central regulatory pathways involved in blood pressure regulation. Leptin plays an important role in the central control of appetite, is also involved in activation of efferent sympathetic pathways to both thermogenic and non‐thermogenic tissues, such as the kidney, and is therefore implicated in obesity‐related hypertension. Leptin is also thought to have a neurotrophic role in the development of the hypothalamus, and altered neonatal leptin profiles secondary to maternal obesity are associated with permanently altered hypothalamic structure and function. In rodent studies, maternal obesity confers persistent sympathoexcitatory hyper‐responsiveness and hypertension acquired in the early stages of development. Experimental neonatal hyperleptinaemia in naive rat pups provides further evidence of heightened sympathetic tone and proof of principle that hyperleptinaemia during a critical window of hypothalamic development may directly lead to adulthood hypertension. Insight from these animal models raises the possibility that early‐life exposure to leptin in humans may lead to early onset essential hypertension. Ongoing mother–child cohort and intervention studies in obese pregnant women provide a unique opportunity to address associations between maternal obesity and offspring cardiovascular function. The goal of the review is to highlight the potential importance of leptin in the developmental programming of hypertension in obese pregnancy.

Sucrose feeding in mouse pregnancy leads to hypertension, and sex-linked obesity and insulin resistance in female offspring

Eating an unbalanced diet during pregnancy may induce long-term health consequences in offspring, in particular obesity, insulin resistance, and hypertension. We tested the hypothesis that a maternal diet rich in simple sugars predispose mouse offspring to obesity, glucose intolerance, and cardiovascular diseases in adulthood. Female C57BL/6J mice were fed either a standard chow or a sucrose-rich diet (26% of total energy) 6 weeks prior to mating, throughout pregnancy and lactation. Offspring of control dams (OC) and high sucrose fed dams (OSF) were weaned onto standard control chow, and metabolic and cardiovascular parameters determined at 3 months of age. Both male and female OSF were hyperphagic by 4 weeks of age and females were heavier than OC at 6 weeks. At 3 months, female OSF showed a significant increase in inguinal fat pad mass, whereas skeletal muscle mass (tibialis anterior) and locomotor activity were decreased relative to OC. A 10-fold increase in fasting serum insulin in female OSF vs. OC at 3 months (Insulin [pmol/L] mean ± SEM, OSF, 200.3 ± 16.1, vs. OC, 20.3 ± 1.8, n = 6 P < 0.001), was associated with impaired glucose tolerance (AUC [mmol/L min] mean ± SEM, OSF 1437.4 ± 124.2 vs. OC, 1076.8 ± 83.9, n = 6, P < 0.05). Both male and female OSF were hypertensive as assessed by radiotelemetry (night-time systolic arterial pressure (SAP) [mmHg] mean ± SEM, male OSF, 128 ± 1 vs. OC, 109 ± 1, n = 6, P < 0.01; female OSF, 130 ± 1 vs. OC, 118 ± 1, n = 6, P < 0.05). Analysis of heart rate variability (HRV) demonstrated an increased low:high frequency ratio in male and female OSF (P < 0.05), indicative of heightened sympathetic efferent tone. Renal tissue noradrenaline (NA) content was markedly raised in the OSF vs. OC (NA [pg/ml/mg tissue] mean ± SEM, male OSF, 2.28 ± 0.19 vs. OC 0.84 ± 0.09, n = 6, P < 0.01). Exposure to a maternal diet rich in sucrose led to obesity and glucose intolerance in female mice offspring, and hypertension in both sexes.

Fostering in mice induces cardiovascular and metabolic dysfunction in adulthood

Non‐technical summary  Cross‐fostering of newborn pups to different dams is a method widely used in rodent studies of developmental ‘programming’ to determine whether pregnancy or the suckling period is more important in determining adult characteristics following changes to the maternal environment. We have investigated whether the process of fostering per se influences cardiovascular and metabolic development in mice. Compared with mice reared by their biological mother, fostered mice showed increased appetite, body weight, abdominal fatness and altered blood sugar metabolism. A marked increase in blood pressure was also apparent. This study demonstrates that the process of fostering can lead to profound effects in cardiovascular and metabolic function in otherwise normal mice. The findings have implications both for the interpretation of previous cross‐fostering studies in mice and for studies investigating the hypothesis of developmental programming, in which early postnatal manipulation of litters is common practice.

Experimental hyperleptinemia in neonatal rats leads to selective leptin responsiveness, hypertension, and altered myocardial function

The prevalence of obesity among pregnant women is increasing. Evidence from human cohort studies and experimental animals suggests that offspring cardiovascular and metabolic function is compromised through early life exposure to maternal obesity. Previously, we reported that juvenile offspring of obese rats develop sympathetically mediated hypertension associated with neonatal hyperleptinemia. We have now addressed the hypothesis that neonatal exposure to raised leptin in the immediate postnatal period plays a causal role. Pups from lean Sprague-Dawley rats were treated either with leptin (3 mg/kg IP) or with saline twice daily from postnatal day 9 to 15 to mimic the exaggerated postnatal leptin surge observed in offspring of obese dams. Cardiovascular function was assessed by radiotelemetry at 30 days, and 2 and 12 months. In juvenile (30 days) leptin-treated rats, hearts were heavier and night-time (active period) systolic blood pressure was raised (mm Hg; mean±SEM: male leptin-treated, 132±1 versus saline-treated, 119±1, n=6, P<0.05; female leptin-treated, 132±2 versus saline-treated, 119±1, n=6, P<0.01), and the pressor response to restraint stress and leptin challenge increased compared with saline-treated rats. Heart rate variability demonstrated an increased low:high frequency ratio in 30-day leptin-treated animals, indicative of heightened sympathetic efferent tone. Echocardiography showed altered left ventricular structure and systolic function in 30-day female leptin versus saline-treated rats. These disorders persisted to adulthood. In isolated hearts, contractile function was impaired at 5 months in male leptin-treated rats. Exogenously imposed hyperleptinemia in neonatal rats permanently influences blood pressure and cardiac structure and function.