Prenatal Tobacco Exposure and Mechanisms of Offspring Obesity

Maternal smoking increases offspring risk for a number of diseases including pediatric and adulthood obesity and type 2 diabetes. Yet, the mechanisms that contribute to the increased obesity and diabetes rates are unknown. Surprisingly, the rates of smoking during pregnancy over the last 12 years have remained unchanged at 15-18%, with regional and demographic variation, despite the well-known negative health outcomes of smoking on offspring health. In a pilot study at the University of Kentucky Medical Center, we found that >30% of the women that delivered had continued to actively smoke during their pregnancy, yielding a large at-risk population, and an opportunity to identify novel mechanisms that contribute to pediatric obesity because of the harmful effects of tobacco smoke exposure during pregnancy. From 47 male neonates, we collected and measured mRNA expression levels in foreskin after circumcision. The gene expression data are suggestive of reduced insulin signaling and increased adipogenesis in infants born to smoking moms. Importantly, we found that epigenetic regulation is likely central to detrimental offspring outcomes as the DNA methylation pattern of at least one key metabolic gene, chemerin, was also significantly altered by maternal smoke exposure. Further, our lab has demonstrated that primary dermal fibroblasts, derived from the same embryonal mesodermal origin as adipocytes, can be isolated to provide a living cell culture system for functional analyses such as glucose uptake and adipogenesis experiments. Notably, we found that cells isolated from babies born to smokers take up more lipid and have decreased glucose uptake because of impaired insulin signaling compared to those cells collected from babies born to nonsmokers. To our knowledge these data are the first to examine signaling pathways to explain how in utero smoke exposure increases adult offspring risk of obesity and diabetes in humans. Our initial goal in the much larger R01 application was to use neonatal tissue to elucidate the potential mechanisms that contribute to increased pediatric obesity and diabetes risks in exposed babies. Our strong preliminary data helped form the basis of our hypotheses that 1) expression as well as DNA methylation of key energy metabolism genes and resulting proteins will be significantly altered in the neonatal tissue of babies born to smokers compared to those born to nonsmokers, 2) living cells isolated from babies born to smokers will be functionally dysregulated compared to cells collected from babies of nonsmokers, and 3) gene and protein expression as well as DNA methylation changes will be identified that are predictive of increased growth and pediatric obesity that can then be used to drive further more detailed mechanistic studies in animal models and primary neonatal fibroblasts. To examine these hypotheses, we planned to recruit a large cohort of pregnant mothers and their male infants and obtain fresh foreskin samples following circumcision. Specific Aim 1 set out to establish potential mechanisms contributing to offspring obesity risk resulting from in utero cigarette exposure. Specific Aim 2 would determine the extent to which living cells are uniquely programmed and functionally dysregulated by in utero tobacco exposure. Meanwhile, Specific Aim 3 would investigate key pathways that lead to obesity susceptibility in 3 year old infants that were exposed to tobacco smoke in utero. As part of the revised Aims for the R56, the scope has been modified in response to reviewer criticisms and will allow the work to fit within a 1 year timeframe with lower available funds. Modified Aim 1 will investigate whether mesenchymal stromal cells (MSCs) isolated from the umbilical cord tissue/blood show similar characteristics (higher chemerin expression, increased adipogenesis) to primary fibroblasts that are isolated from the foreskin. This is important for two reasons: (1) MSCs can directly differentiate into adipocytes in humans unlike primary dermal fibroblasts, making them more physiologically relevant; and (2) MSCs could be isolated in the future from both boys and girls as a major criticism of our work is that we can only do our experiments in male neonates. Modified Aim 2 will continue our focus on the potential mechanism for increased obesity risk in offspring. We identified an epigenetically controlled gene, chemerin, in human samples that may be responsible, and we plan to measure chemerin protein and gene expression levels in mouse offspring born to vehicle or smoke exposed dams. These samples are already collected so no live animal studies will be performed as part of the R56. However, it is critical to identify whether offspring exposed to tobacco smoke, that are predisposed to obesity, have higher chemerin levels. These experiments will provide further evidence so we can move from the bedside back to the bench and test this critical pathway associated with obesity susceptibility using genetic manipulation of chemerin and/or its receptor in mouse lines or in primary fibroblasts (gene knockout or overexpression) in a future grant application. Overall Impact: Research findings using tissues/cells from babies born to smokers can provide an innovative approach and proof of concept to understand the effects of various other in utero environmental exposures on metabolic outcomes in children. Future work can also apply this model to investigate new preventative and intervention approaches targeting identified pathways.