Paternal Contributions to Metabolic Disease in Offspring: Environment, Epigenetics, and Sperm

项目概要 一种减轻 2 型糖尿病和肥胖负担的新方法来自于认识到父母 肥胖或糖尿病可通过非遗传效应增加后代患代谢疾病的风险。这个观点 提出了一个令人兴奋的可能性，即在受孕前对父母进行改善新陈代谢的治疗可以 中断肥胖和糖尿病的恶性代际循环，从而改善后代的健康。 在之前的资助周期中，我们开发了以父亲为重点的干预措施对精子影响的证据 表观基因组和后代健康。我们最近在人类身上证明，父亲肥胖与 后代脐带血中 DNA 甲基化发生改变7。我们在小鼠身上的新数据表明 (1) 父亲肥胖和 高血糖是后代健康的关键决定因素，(2) 改善父亲的新陈代谢 - 通过治疗 SGLT2 抑制剂或热量限制 - 可以逆转与年龄相关的体重增加、胰岛素抵抗、血糖 F1 后代的不耐受和脂肪肝，(3) 父本代谢有效改变胎儿的转录组 和成年后代，包括氧化和脂质调节基因，以及（4）父本代谢改变 精子和后代组织中的表观基因组。例如，增强子处的 DNA 羟甲基化 (5hmC) 在 HFD 喂养的雄性精子中，与差异表达基因相邻的基因减少，而在 用 SGLT2i 或 AMP 激酶激活剂 AICAR 治疗的男性。 在这个修订后的应用程序中，我们将利用这种创新的小鼠模型来确定 父亲的健康及其治疗调节父亲的生殖细胞表观基因组，以改善后代的健康，并 检验 AMPK-Tet2 通路（葡萄糖介导的表观遗传调控的介质）的假设， 介导观察到的精子 5hmC 差异和后代代谢疾病。在目标 1 中，我们将定义 父亲肥胖和高血糖的影响及其逆转对生殖细胞表观基因组的影响（5mC/5hmC，小 RNA），并识别关键的顺式调节元件。在目标 2 中，我们将确定生殖细胞的致病性 通过评估体外受精和精子衍生的 ncRNA 后的后代结果来观察表观遗传变化 注射。目标 3 将确定父本对后代的影响是否是通过对胚胎的直接影响来介导的， 或影响胎盘发育的胚胎外谱系，使用单细胞转录组分析 单细胞水平的囊胚以及来自对照、HFD 和胎盘的详细形态分析 接受 HFD CANA 治疗的父亲。目标 4 将检验以下假设：AMPK-Tet2 途径是葡萄糖- 介导体细胞的表观遗传调控，还介导葡萄糖诱导的生殖细胞变化 表观基因组。我们将分析体内 AMPK 激活对野生型或 Tet2-null 精子中 5hmC 的影响 小鼠及其后代表型。总之，识别父系反应的因果机制 高血糖和肥胖及其逆转将为指导新药物的开发提供重要的新信息 以父亲为中心的可转化方法，以减轻子孙后代的疾病负担。

PROJECT SUMMARY One novel approach to reduce the burden of type 2 diabetes and obesity comes from the recognition that parental obesity or diabetes can increase risk of metabolic disease in offspring via non-genetic effects. This perspective raises the exciting possibility that treatment of parents to improve their metabolism before conception could interrupt vicious intergenerational cycles of obesity and diabetes, thus improving offspring health. In the prior funding cycle, we developed evidence for the impact of paternally-focused interventions on the sperm epigenome and offspring health. We recently demonstrated in humans that paternal obesity is associated with altered DNA methylation in cord blood of offspring7. Our new data in mice indicate (1) paternal obesity and hyperglycemia are key determinants of offspring health, (2) improving paternal metabolism - by treatment with either SGLT2 inhibitors or caloric restriction - can reverse age-associated weight gain, insulin resistance, glucose intolerance, and fatty liver in F1 offspring, (3) paternal metabolism potently modifies the transcriptome in fetal and adult offspring, including oxidative and lipid regulatory genes, and (4) paternal metabolism alters the epigenome in both sperm and offspring tissue. For example, DNA hydroxymethylation (5hmC) at enhancers adjacent to differentially expressed genes is reduced in sperm of HFD-fed males, and reciprocally increased in males treated with SGLT2i or the AMP kinase activator AICAR. In this revised application, we will utilize this innovative mouse model to identify the mechanisms by which paternal health and its treatment modulate the paternal germ cell epigenome to improve offspring health, and to test the hypothesis that the AMPK-Tet2 pathway, a mediator of glucose-mediated epigenetic regulation, mediates observed differences in sperm 5hmC and offspring metabolic disease. In Aim 1, we will define the impact of paternal obesity and hyperglycemia, and its reversal, on the germ cell epigenome (5mC/5hmC, small RNA), and identify key cis-regulating elements. In Aim 2, we will determine the pathogenicity of germ cell epigenetic changes by evaluating offspring outcomes after in vitro fertilization and sperm-derived ncRNA injection. Aim 3 will determine whether paternal effects on offspring are mediated via direct effects on the embryo, or on extraembryonic lineages affecting placental development, using single-cell transcriptomic analysis of blastocysts at a single-cell level and detailed morphometric analysis of placentae derived from control, HFD, and HFD+CANA-treated fathers. Aim 4 will test the hypothesis that the AMPK-Tet2 pathway, a mediator of glucose- mediated epigenetic regulation in somatic cells, also mediates glucose-induced changes in the germ cell epigenome. We will analyze the impact of AMPK activation in vivo on 5hmC in sperm from wild type or Tet2-null mice and on offspring phenotypes. In summary, identification of causal mechanisms in response to paternal hyperglycemia and obesity - and its reversal - will provide critical new information to guide development of new paternally-focused translatable approaches to reduce disease burden in future generations.