M. Renee Prater

Current Thoughts on Maternal Nutrition and Fetal Programming of the Metabolic Syndrome

Chronic diseases such as type 2 diabetes and cardiovascular disease are the leading cause of death and disability worldwide. Although the metabolic syndrome has been defined in various ways, the ultimate importance of recognizing this combination of disorders is that it helps identify individuals at high risk for both type 2 diabetes and cardiovascular disease. Evidence from observational and experimental studies links adverse exposures in early life, particularly relating to nutrition, to chronic disease susceptibility in adulthood. Such studies provide the foundation and framework for the relatively new field of developmental origins of health and disease (DOHaD). Although great strides have been made in identifying the putative concepts and mechanisms relating specific exposures in early life to the risk of developing chronic diseases in adulthood, a complete picture remains obscure. To date, the main focus of the field has been on perinatal undernutrition and specific nutrient deficiencies; however, the current global health crisis of overweight and obesity demands that perinatal overnutrition and specific nutrient excesses be examined. This paper assembles current thoughts on the concepts and mechanisms behind the DOHaD as they relate to maternal nutrition, and highlights specific contributions made by macro- and micronutrients.

Intrauterine exposure to high saturated fat diet elevates risk of adult-onset chronic diseases in C57BL/6 mice.

BACKGROUND The developmental environment is thought to determine, in part, lifelong metabolic parameters and risk of adult disease. Effects of maternal malnutrition on fetal growth have been studied extensively, and the role of poor prenatal diet in elevating lifelong risk of cardiovascular and metabolic disease has been well characterized (www.thebarkertheory.com). However, the contribution of gestational high saturated fat diet (HFD) to adult-onset metabolic disease and skeletal dysfunction has only recently been recognized, and as such is incompletely understood. METHODS The present study evaluates the pathophysiologic mechanisms linking gestational HFD (approximating the macronutrient content of fast food) and elevated oxidative stress (OS) to adult-onset skeletal, cardiovascular, and metabolic dysfunction. RESULTS Results of this study demonstrate that adult offspring of dams fed HFD during pregnancy exhibited adult hyperglycemia, insulin resistance, obesity, and hypertension, despite being fed healthy standard rodent chow throughout postnatal life. These offspring also showed significantly lower femoral epiphyseal average bone mineral density (ABMD) at 6 months of age, and dysregulation of distal femoral trabecular architecture at 12 months of age, characteristic of osteoporosis. Incidence of these adult-onset adverse skeletal and metabolic effects was reduced by supplementing the pregnant dam with the antioxidant (quercetin, Q) during pregnancy. CONCLUSIONS Collectively, these data suggest that offspring of dams who consume a diet rich in saturated fats during pregnancy are at increased risk of adult-onset chronic disease. Additionally, these chronic diseases were determined to be in-part OS-mediated, and preventable by increasing a prenatal dietary antioxidant; this knowledge offers both a putative mechanism of disease pathogenesis and suggests a potential preventive strategy.

Micro-CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear-staining methods.

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear-staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro-CT) imaging is a versatile, well-documented tool traditionally used to generate high-resolution 3-D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro-CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro-CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro-CT measurement of fetal limb lengths correlates well with traditional clear-staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro-CT and 10.82% for clear-staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro-CT technique to generate 3-D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear-staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well.

Gestational high saturated fat diet alters C57BL/6 mouse perinatal skeletal formation

BACKGROUND Our present work joins growing evidence that gestational environment (maternal nutrition, health, and chemical exposures) strongly influences prenatal development (www.thebarkertheory.org). The present study suggests that maternal consumption of a diet high in saturated fats (HFD), which approximates the macronutrient content of fast food, impairs perinatal skeletal development. METHODS In this study, administration of HFD (32% saturated fat) for one month prior to conception and throughout gestation in C57BL/6J mice was associated with a marked reduction in late-gestation fetal skeletal developmental delay that included shorter long bone lengths, decreased average bone mineral density (ABMD; 20%), lower total bone volume (TBV; 45%), and shorter crown-to-rump length (C-R; 12%), as compared to controls. RESULTS A putative mechanism linking prenatal HFD to dysregulated fetal osteogenesis is HFD-induced oxidative stress (OS), which has been shown in our laboratory to cause placental labyrinthine vascular damage and impaired fetal signaling pathways associated with osteogenesis (Liang et al., unpublished data). CONCLUSIONS The theory of HFD-associated, OS-mediated placental damage and skeletal pathogenesis was supported by demonstrating a protective effect of the dietary antioxidant quercetin (Q) against HFD-associated fetal skeletal developmental delay. Improved understanding of the role of HFD and elevated OS in fetal skeletal development will help to more completely elucidate the importance of the prenatal environment to fetal formation, and will be applied to better understand the contribution of the fetal environment to long-term risk of adult-onset disease.

Mitigating or Exacerbating Effects of Maternal-Fetal Programming of Female Mice Through the Food Choice Environment

Humans live, eat, and become overweight/obese in complex surroundings where there are many available food choices. Prenatal exposure to poor food choices predisposes offspring to increased negative health risks, including obesity. Many animal experiments have analyzed intergenerational body weight parameters in an environment without food choices, which may not be directly translatable to the human food environment. In this study, offspring from mothers with a defined high-fat diet (HFD) or low-fat diet (LFD) were arbitrarily assigned to either an exclusively LFD or HFD or to a diet where they have a choice between LFD and HFD (choice diet). Offspring displayed negative outcomes of increased body weight, body fat, serum leptin, and blood glucose levels when given the choice diet compared with offspring on the LFD. Conversely, improved energy expenditure was found for offspring given the choice diet compared with offspring from HFD dams given LFD. In addition, maternal diet-specific influences on offspring metabolic parameters were identified, especially in offspring from HFD dams, including positive outcomes of reduced leptin in LFD offspring, reduced corticosterone and cholesterol levels in HFD offspring, and increased exercise levels in choice offspring, as well as the negative outcome of increased calorie intake in LFD offspring from HFD dams. This defined model can now be used as the basis for future studies to characterize the cycle of inter- and intragenerational obesity and whether more realistic diet environments, especially those including choice, can mitigate phenotype.

Late-gestation ventricular myocardial reduction in fetuses of hyperglycemic CD1 mice is associated with increased apoptosis

BACKGROUND Previous work in our laboratory showed reduced myocardium and dilated ventricular chambers in gestation day (GD) 17 hearts that were collected from hyperglycemic CD1 mouse dams. Pre-breeding maternal immune stimulation, using Freunds complete adjuvant (FCA), diminished the severity of these fetal heart lesions. The following experiments were performed to detect possible changes in fetal heart apoptotic cell death, under hyperglycemic conditions and with or without maternal immune stimulation. METHODS Female CD1 mice were injected with 200 mg/kg of streptozocin (STZ) to induce insulin-dependent diabetes mellitus. Half of these mice received prior FCA injection. Fetal hearts were collected on GD 17 and myocardial apoptotic cells were quantified using flow cytometry. A panel of apoptosis regulatory genes (Bcl2, p53, Casp3, Casp9, PkCe) was then examined in the fetal myocardium using RT-PCR. RESULTS Early apoptotic cells and late apoptotic/necrotic cells were significantly increased in fetal hearts from STZ or STZ+FCA dams. Pre-treatment with FCA reduced late apoptotic/necrotic cells to control level, suggesting some cell death protection was rendered by FCA. Paradoxically in the face of such increased cell death, the expression of pro-apoptotic genes Casp3 and Casp9 was decreased by diabetes, while the anti-apoptotic gene Bcl2 was increased. CONCLUSIONS Maternal hyperglycemia causes dys-regulated apoptosis of fetal myocardial cells. Such effect may be prevented by maternal immune stimulation.

Gold Nanoparticle Toxicity in Mice and Rats: Species Differences:

Nanotoxicity studies are greatly needed to advance nanomedical technologies into clinical practice. We assessed the toxic effects of a single intravenous exposure to commercially available gold nanoparticles (GNPs) in mice and rats. Fifteen-nm GNPs were purchased and independently characterized. Animals were exposed to either 1,000 mg GNPs/kg body weight (GNP group) or phosphate-buffered saline. Subsets of animals were euthanized and samples collected at 1, 7, 14, 21, and 28 days postexposure. Independent characterization demonstrated that the physicochemical properties of the purchased GNPs were in good agreement with the information provided by the supplier. Mice exposed to GNPs developed granulomas in the liver and transiently increased serum levels of the pro-inflammatory cytokine interleukin-18. No such alterations were found in rats. While there was no fatality in mice post-GNP exposure, a number of the rats died within hours of GNP administration. Differences in GNP biodistribution and excretion were also detected between the two species, with rats having a higher relative accumulation of GNPs in spleen and greater fecal excretion. In conclusion, GNPs have the ability to incite a robust macrophage response in mice, and there are important species-specific differences in their biodistribution, excretion, and potential for toxicity.