Maternal obesity increases the risk of cardiovascular and metabolic disease in the offspring both during childhood and adult life. Pregnant women and mice with obesity have lower circulating levels of adiponectin (ADN) compared to lean controls. ADN is an adipokine involved in regulating energy metabolism, vascular function, and placental function. We hypothesized that offspring of obese mice have impaired resistance artery function, which can be prevented by restoration of normal circulating ADN levels in obese dams during late pregnancy. Adult female mice were fed either control or obesogenic diet and mated with control diet-fed males. Control dams received a continuous infusion of phosphate saline buffer (PBS) during late pregnancy whereas obese females received either PBS or ADN. After weaning, offspring were fed a control diet. Mesenteric arteries (MsA) were dissected from adult offspring and mounted in a wire myograph or fixed for histology. MsA responses to vasoconstrictors (phenylephrine and endothelin-1) were not different between infusion groups. However, the vasodilatory responses to acetylcholine were reduced in offspring from obese dams as compared to control-fed dams. ADN supplementation during pregnancy restored the cholinergic vasodilatory responses of resistance vessels in offspring from obese dams. These observations suggest that normalizing circulating adiponectin levels in pregnancies complicated by obesity prevents in utero programming of vascular dysfunction in the offspring.

Maternal obesity in pregnancy is strongly associated with complications such as fetal overgrowth and infants of obese mothers have an increased risk to develop obesity, diabetes, and cardiovascular disease later in life. However, the underlying mechanisms are not well established. Circulating levels of adiponectin are low in obese pregnant women and maternal circulating adiponectin is negatively associated with birth weight. We have reported that normalizing maternal adiponectin in obese pregnant mice prevents placental dysfunction, fetal overgrowth, and programming of offspring cardio-metabolic disease. However, the mechanistic link between maternal adiponectin, placental function, and fetal growth remains to be established. We hypothesized that trophoblast-specific overexpression of the adiponectin receptor 2 (Adipor2) in healthy pregnant mice inhibits placental mTORC1 signaling and nutrient transport, resulting in fetal growth restriction. Using lentiviral transduction of blastocysts with a mammalian gene expression lentiviral vector for up-regulation of Adipor2 (Adipor2-OX), we achieved a ~ 3-fold increase in placenta Adipor2 mRNA levels and a 2-fold increase of the ADIPOR2 protein in the trophoblast plasma membrane. Placenta-specific Adipor2-OX increased placental peroxisome proliferator-activated receptor-α phosphorylation, ceramide synthase expression and ceramide concentrations. Furthermore, Adipor2-OX inhibited placental mTORC1 signaling and reduced in vivo placental transport of glucose and amino acids. Lastly, Adipor2-OX reduced fetal weight by 11%. These data provide mechanistic evidence that placental Adipor2 signaling directly affects fetal growth. We propose that low circulating adiponectin in maternal obesity causes fetal overgrowth and programs the offspring for cardio-metabolic disease mediated by a direct effect on placental function.

BACKGROUND:Pregnancies complicated by maternal obesity are characterized by metabolic differences affecting placental nutrient transport and fetal development. Docosahexaenoic acid (DHA) is critical for fetal brain development and is primarily incorporated into phosphatidylcholine (PC). Recent evidence suggests that choline may enhance PC-DHA synthesis; however, data on the impact of maternal plasma choline on placental phospholipid DHA content in females with obesity are limited. METHODS:We conducted a secondary analysis of a DHA supplementation trial (800 mg/d) in 38 pregnant females with obesity (body mass index ≥30 kg/m2). Blood samples at 36 wk gestation and term placentas were analyzed for phospholipids using mass spectrometry. Choline transporter-like (CTL) proteins in the syncytiotrophoblast microvillous (MVM) and basal plasma membranes were quantified by Western blot. RESULTS:Daily DHA supplementation from 25 wk gestation was associated with higher maternal plasma and placental PC- and lysophosphatidylcholine (LPC)-DHA. A significant interaction (P interaction <0.05) between DHA supplementation and choline indicated that higher choline enhanced the incorporation of DHA into plasma PC. MVM CTL-1 expression was correlated with placental total PC-DHA and LPC-DHA content, suggesting that CTL-1 has a predominate role in placental choline uptake and phospholipid synthesis. CONCLUSIONS:These findings suggest that choline may influence maternal PC- and LPC-DHA synthesis and plasma levels, as well as the expression of placental choline transporters and the resulting PC- and LPC-DHA content in females with obesity. These relationships may have implications for DHA transport to the fetus and overall fetal development.

Pregnant women with obesity are more likely to deliver infants who are large for gestational age (LGA). LGA is associated with increased perinatal morbidity and risk of developing metabolic disease later in life. However, the mechanisms underpinning fetal overgrowth remain to be fully established. Here, we identified maternal, placental, and fetal factors that are associated with fetal overgrowth in pregnant women with obesity. Maternal and umbilical cord plasma and placentas were collected from women with obesity delivering infants who were LGA (n=30) or appropriate for gestational age (AGA, n=21) at term. Maternal and umbilical cord plasma analytes were measured using multiplex sandwich assay and ELISA. Insulin/mechanistic target of rapamycin (mTOR) signaling activity was determined in placental homogenates. Amino acid transporter activity was measured in isolated syncytiotrophoblast microvillous membrane (MVM) and basal membrane (BM). Glucagon-like peptide-1 receptor (GLP-1R) protein expression and signaling were measured in cultured primary human trophoblast (PHT) cells. Maternal plasma glucagon-like peptide-1 (GLP-1) was higher in LGA pregnancies and positively correlated to birthweight. Umbilical cord plasma insulin, C-peptide, and GLP-1 were increased in obese-large for gestational age (OB-LGA) infants. LGA placentas were larger but showed no change in insulin/mTOR signaling or amino acid transport activity. GLP-1R protein was expressed in the MVM isolated from human placenta. GLP-1R activation stimulated protein kinase alpha (PKA), extracellular signal-regulated kinase-1 and-2 (ERK1/2), and mTOR pathways in PHT cells. Our results suggest elevated maternal GLP-1 may drive fetal overgrowth in obese pregnant women. We speculate that maternal GLP-1 acts as a novel regulator of fetal growth by promoting placental growth and function.

Infants born to obese mothers have a greater risk for childhood obesity and insulin resistance. However, the underlying biological mechanism remains elusive, which constitutes a significant roadblock for developing specific prevention strategies. Maternal adiponectin levels are lower in obese pregnant women, which is linked with increased placental nutrient transport and fetal overgrowth. We have previously reported that adiponectin supplementation to obese dams during the last four days of pregnancy prevented the development of obesity, glucose intolerance, muscle insulin resistance, and fatty liver in three months old offspring. In the present study, we tested the hypothesis that 6-9-month-old offspring of obese dams show glucose intolerance associated with muscle insulin resistance and mitochondrial dysfunction and that normalization of maternal adiponectin in obese pregnant mice prevents the development of this phenotype in the offspring. Male and female offspring of obese mice exhibited in vivo glucose intolerance and insulin resistance at 6 and 9 months of age. In gastrocnemius muscles ex vivo, male and female offspring of obese dams showed reduced phosphorylation of insulin receptor substrate 1Tyr-608 , AktThr-308 , and decreased Glut4 plasma membrane translocation upon insulin stimulation. These metabolic abnormalities in offspring born to obese mice were largely prevented by normalization of maternal adiponectin levels in late pregnancy. We provide evidence that low circulating maternal adiponectin is a critical mechanistic link between maternal obesity and the development of metabolic disease in offspring. Strategies aimed at improving maternal adiponectin levels may prevent long-term metabolic dysfunction in offspring of obese mothers.

Maternal hypercholesterolemia (MHC), a pathological condition characterized by an exaggerated rise in maternal serum cholesterol during pregnancy, may influence offspring hepatic lipid metabolism and increase the risk of nonalcoholic fatty liver disease (NAFLD). As NAFLD is characterized by a sexual dimorphic response, we assessed whether early-life exposure to excessive cholesterol influences the development of NAFLD in offspring and whether this occurs in a sex-specific manner. Female apoE−/− mice were randomly assigned to a control (CON) or a high cholesterol (CH; 0.15%) diet prior to breeding. At parturition, a cross-fostering approach was used to establish three groups: (1) normal cholesterol exposure throughout gestation and lactation (CON-CON); (2) excessive cholesterol exposure throughout gestation and lactation (CH-CH); and (3) excessive cholesterol exposure in the gestation period only (CH-CON). Adult male offspring (PND 84) exposed to excessive cholesterol during gestation only (CH-CON) demonstrated hepatic triglyceride (TG) accumulation and reduced lipogenic gene expression. However, male mice with a prolonged cholesterol exposure throughout gestation and lactation (CH-CH) had a similar, but not exacerbated hepatic response. Further, with the exception of higher serum TG in adult CH-CH females, evidence for a programming effect in female offspring was largely absent in comparison with males. These results indicate a sexual dimorphic response with respect to the effect of MHC on later life hepatic steatosis and highlight the gestation period as the most influential malprogramming window for hepatic lipid dysfunction in males.

Placental regulation of fetal growth involves the integration of multiple signaling pathways that modulate an array of placental functions, including nutrient transport. As a result, the flux of oxygen and nutrients to the fetus is altered, leading to changes in placental and fetal growth. Placental insulin/insulinlike growth factor-1 and mechanistic target of rapamycin signaling and amino acid transport capacity are inhibited in fetal growth restriction and activated in fetal overgrowth, implicating these placental functions in driving fetal growth. With novel approaches to specifically target the placenta, clinical interventions to modulate placental function in high-risk pregnancies can be developed.

As a collection of metabolic abnormalities including inflammation, insulin resistance, hypertension, hormone imbalance, and dyslipidemia, maternal obesity has been well-documented to program disease risk in adult offspring. Although hypercholesterolemia is strongly associated with obesity, less work has examined the programming influence of maternal hypercholesterolemia (MHC) independent of maternal obesity or high-fat feeding. This study was conducted to characterize how MHC per se impacts lipid metabolism in offspring. Female ( n = 6/group) C57BL/6J mice were randomly assigned to: (1.) a standard chow diet (Control, CON) or (2.) the CON diet supplemented with exogenous cholesterol (CH) (0.15%, w/w) throughout mating and the gestation and lactation periods. At weaning (postnatal day (PND) 21) and adulthood (PND 84), male offspring were characterized for blood lipid and lipoprotein profile and hepatic lipid endpoints, namely cholesterol and triglyceride (TG) accumulation, fatty acid profile, TG production, and mRNA expression of lipid-regulatory genes. Both newly weaned and adult offspring from CH mothers demonstrated increased very low-density lipoprotein (VLDL) particle number and size and hepatic TG and n-6 polyunsaturated fatty acid accumulation. Further, adult CH offspring exhibited reduced fatty acid synthase ( Fasn ) and increased diglyceride acyltransferase ( Dgat1 ) mRNA expression. These programming effects appear to be independent of changes in hepatic TG production and postprandial lipid clearance. Study results suggest that MHC, independent of obesity or high-fat feeding, can induce early changes to serum VLDL distribution and hepatic lipid profile that persist into adulthood.

The in utero and immediate postnatal environments are recognized as critical windows of developmental plasticity where offspring are highly susceptible to changes in the maternal metabolic milieu. Maternal hypercholesterolemia (MHC) is a pathological condition characterized by an exaggerated rise in maternal serum cholesterol during pregnancy which can program metabolic dysfunction in offspring, including dysregulation of hepatic lipid metabolism. Although there is currently no established reference range MHC, a loosely defined cutoff point for total cholesterol >280 mg/dL in the third trimester has been suggested. There are several unanswered questions regarding this condition particularly with regard to how the timing of cholesterol exposure influences hepatic lipid dysfunction and the mechanisms through which these adaptations manifest in adulthood. Gestational hypercholesterolemia increased fetal hepatic lipid concentrations and altered lipid regulatory mRNA and protein content. These early changes in hepatic lipid metabolism are evident in the postweaning environment and persist into adulthood. Further, changes to hepatic epigenetic signatures including microRNA (miR) and DNA methylation are observed in utero, at weaning, and are evident in adult offspring. In conclusion, early exposure to cholesterol during critical developmental periods can predispose offspring to the early development of nonalcoholic fatty liver disease (NAFLD) which is characterized by altered regulatory function beginning in utero and persisting throughout the life cycle.

Supported by the National Institute for Complementary and Alternative Medicine.

Upon completion, participant will be able to assess the risks of hypercholesterolemia during pregnancy.

Maternal hypercholesterolemia (MHC) is a pathological condition characterized by an exaggerated rise in maternal serum cholesterol during gestation, which can alter offspring hepatic lipid metabolism. However, the extent that these maladaptations occur during gestation and the molecular mechanisms involved remain unknown. MicoRNAs (miRNA) are small, noncoding RNAs that contribute to the development and progression of nonalcoholic fatty liver disease. Therefore, we sought to determine the degree to which in utero exposure to excessive cholesterol affects fetal hepatic lipid metabolism and miRNA expression. Twelve female apoE(-/-) mice were randomly assigned to two different chow-based diets throughout gestation: control (CON) or the CON diet with cholesterol (0.15%). MHC reduced maternal fecundity and reduced litter size and weight. On gestational day 18, fetuses from MHC dams possessed increased placental cholesterol and hepatic triglycerides (TG), which were accompanied by a downregulation in the expression of hepatic lipogenic and TG synthesis and transport genes. Furthermore, fetal livers from MHC mothers showed increased miRNA-27a and reduced miRNA-200c expression. In summary, in utero exposure to MHC alters fetal lipid metabolism and lends mechanistic insight that implicates early changes in miRNA expression that may link to later-life programming of disease risk.

SCOPE:The programming of hepatic lipid dysfunction in response to early cholesterol exposure and the influencing effects of postnatal diet is evaluated in apoE-/- mice. METHODS AND RESULTS:In two separate studies, female mice are assigned to a standard chow (S) or a cholesterol-enriched chow (C) diet during gestation and lactation. Male offspring from each dam are weaned on a postnatal S or a hypercaloric western (W) diet resulting in four experimental groups: S-S and C-S (Experiment 1) and S-W and C-W (Experiment 2). At weaning, litters from hypercholesterolemic mothers weighed less (p < 0.05) and pups had higher blood lipids, glucose, and hepatic cholesterol compared with pups from S-fed mothers. Adult C-S offspring demonstrate an atherogenic lipid profile and increased (p < 0.05) hepatic cholesterol and triglyceride content with altered lipid regulatory mRNA expression and protein content compared with S-S offspring. Alternatively, no difference (p > 0.05) is observed between S-W and C-W in serum and hepatic lipid profiles; however, serum AST and ALT are higher (p < 0.05) in C-W versus S-W offspring. CONCLUSION:The degree of hepatic lipid deposition observed in adult offspring exposed to excessive early cholesterol is influenced by the postnatal diet.

During hypercholesterolemic pregnancies, the fetus is exposed to excessive cholesterol that is thought to increase cardiovascular disease risk in adults. We recently reported that newly‐weaned offspring from hypercholesterolemic apoE−/− mice have increased blood lipid concentrations that are normalized by maternal phytosterol (PS) supplementation throughout gestation and lactation. However, the influence of maternal hypercholesterolemia and PS supplementation on offspring oxysterols, bioactive oxygenated cholesterol derivatives that regulate arterial health and lesion progression, have yet to be addressed. Therefore, we tested the hypothesis that offspring born to hypercholesterolemic mothers would have increased circulating oxysterol concentrations and that pups from PS‐supplemented mothers would have a more favorable serum and hepatic oxysterol profile compared with pups from unsupplemented mothers. Twenty‐one female apoE−/− mice were randomly assigned to three different chow based diets throughout gestation and lactation: chow (chow; control), chow diet with high cholesterol (CH; 0.15% (w/w), and chow diet with 0.15% (w/w) cholesterol and 2% (w/w) phytosterol (CH/PS). On postnatal day 21, offspring were euthanized for serum and hepatic oxysterol profiling and transcriptional changes in hepatic sterol regulatory targets by HPLC and RT‐PCR, respectively. Pups born to hypercholesterolemic mothers had increased (p<0.05) serum cholesterol, 25‐hydroxycholesterol (HC), 7αHC, 7βHC, and 7‐ketocholesterol that were normalized by maternal PS‐supplementation. Hepatic mRNA expression of 7α‐hydroxylase (CYP7A1) was increased (p<0.05) in pups born to hypercholesterolemic mothers but normalized in pups from PS‐supplemented mothers. Compared with the CH group, pups from PS‐supplemented mothers demonstrated increased transcription of liver X receptor (LXR) and its target gene adenosine triphosphate binding cassette G8 (ABCG8). No difference (p>0.05) in the transcription of hepatic oxysterol binding proteins (OSBP 3, 8, and 7) was observed between the groups. These results suggest that, beyond increased circulating cholesterol, newly‐weaned offspring from hypercholesterolemic mothers have enhanced serum oxysterol concentrations and that maternal PS supplementation attenuates this response.Support or Funding InformationSupported by the National Institute for Complementary and Alternative Medicine.

Early exposure to excessive cholesterol, in utero or in the early postnatal life, may program lipid metabolism in later life and predispose adult offspring to cardiovascular disease risk. However, little is known regarding the influence of maternal hypercholesterolemia on blood lipids and lipoprotein distribution throughout the life course. To better understand the influence of maternal hypercholesterolemia on blood lipids and lipoprotein distribution in offspring, blood lipid phenotyping was performed on newly‐weaned offspring (postnatal day 21) and chow‐fed adult offspring (postnatal day 84) born to mothers fed a chow diet or a chow diet supplemented with 0.15% cholesterol throughout gestation and lactation (n=6/group). Blood lipids including total‐C, HDL‐C, and LDL‐C were measured with enzymatic assay, and blood lipoprotein particle number and size were determined through nuclear magnetic resonance spectroscopy. Maternal serum total‐C during gestation (week 2) was higher (p≤0.05) in cholesterol‐supplemented dams compared with chow‐fed dams (87.5 ± 3.7 vs. 67.0 ± 3.5 mg/dL). Newly‐weaned and adult pups from chow or cholesterol‐supplemented mothers did not differ (p≥0.05) in serum lipid profile (total‐C, LDL‐C, or HDL‐C) or LDL and HDL particle number and size. However, both newly‐weaned pups and adult offspring born to cholesterol‐supplemented mothers demonstrated an increase (p≤0.05) in VLDL particle number and VLDL size compared with offspring from chow‐fed mothers. These results suggest that early exposure to cholesterol through maternal diet‐induced hypercholesterolemia influences VLDL distribution and size in newly‐weaned mouse offspring that persists into adulthood.Support or Funding InformationSupported by the National Institute for Complementary and Alternative Medicine.

The tremendous impact of cardiovascular diseases (CVDs) on the health and well-being of Americans cannot be overemphasized as it presents major challenges to society on several fronts. Dyslipidemia is recognized as a major preventable risk factor in the pathophysiology of CVD. Adding to the urgency of CVD as a global health issue is the rise in dyslipidemic risk factors among women of child-bearing age due to underlying genetic, disease state, and dietary factors. This chapter provides an overview of what is currently known regarding the influence of maternal dyslipidemia on lipid metabolism and CVD risk in offspring and examines the safety and efficacy of natural health product supplementation in obese and dyslipidemic pregnancies to protect the fetus from maladaptive early exposure to dyslipidemic CVD risk factors. Lifestyle therapies, including both dietary and exercise interventions, are considered first-line defenses in reducing maternal dyslipidemia.

In hypercholesterolemic pregnancies, the maternal environment is characterized by excessive levels of atherogenic lipids that may increase cardiovascular disease risk in mothers and their offspring. We examined the influence of maternal hypercholesterolemia and phytosterol (PS) intervention on the concentration and metabolism of oxysterols, bioactive oxygenated cholesterol derivatives that regulate arterial health and lesion progression, in mothers and their newly weaned offspring. Twenty-one female apoE-/- mice were randomly assigned to three different diets throughout gestation and lactation: (1) chow, (2) high cholesterol (CH; 0.15%) and (3) CH with added PS (2%, CH/PS). At the end of the lactation period, mothers and pups were euthanized for serum and hepatic oxysterol analyses, hepatic transcriptional profiling of hepatic sterol regulatory targets and atherosclerosis. Hypercholesterolemic dams and their pups demonstrated increased (P˂.05) serum oxysterols [including 24 hydroxycholesterol (HC), 25HC, 27HC, 7αHC, 7βHC and 7 ketocholesterol)] compared with the chow group that were normalized by maternal PS supplementation. Hepatic oxysterol concentrations followed a similar pattern of response in mothers but were not altered in newly weaned pups. Hepatic mRNA expression suggested a pattern of enhanced abca1/g1 high-density-lipoprotein-mediated efflux but a reduction in biliary abcg5/g8 export in both dams and their pups. Although arterial lesions were not apparent in newly weaned pups, CH dams demonstrated enhanced atherosclerosis that was reduced upon PS intervention. These results demonstrate that offspring from hypercholesterolemic pregnancies have enhanced circulating oxysterol concentrations and highlight the potential utility of PS as a lipid-lowering option during hypercholesterolemic pregnancies for which there are currently limited options.