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

项目摘要 一种新的方法来减少2型糖尿病和肥胖的负担来自于认识到，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。这个角度 提出了一种令人兴奋的可能性，即在怀孕前对父母进行治疗以改善他们的新陈代谢， 打破肥胖和糖尿病的恶性代际循环，从而改善后代的健康。 在上一个资助周期中，我们发现了以父亲为中心的干预对精子影响的证据。 表观基因组和后代健康。我们最近在人类中证明，父亲肥胖与 在后代的脐带血中改变DNA甲基化7.我们在小鼠中的新数据表明（1）父亲肥胖， 高血糖症是后代健康的关键决定因素，（2）改善父亲的新陈代谢-通过用 SGLT 2抑制剂或热量限制-可以逆转年龄相关的体重增加、胰岛素抵抗、葡萄糖 不耐受和脂肪肝的F1后代，（3）父亲的代谢有力地修改转录组在胎儿 和成年后代，包括氧化和脂质调节基因，和（4）父亲的代谢改变了 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（5 hmC） 在HFD喂养的雄性精子中，与差异表达基因相邻的基因减少， 接受SGLT 2 i或AMP激酶激活剂AICAR治疗的雄性。 在这个修改后的应用程序中，我们将利用这种创新的小鼠模型来确定机制， 父亲健康及其治疗调节父亲生殖细胞表观基因组以改善后代健康， 检验AMPK-Tet 2通路（葡萄糖介导的表观遗传调节的介质） 介导精子5 hmC和后代代谢疾病中观察到的差异。在目标1中，我们将定义 父亲肥胖和高血糖及其逆转对生殖细胞表观基因组（5 mC/5 hmC，小 RNA），并确定关键的顺式调节元件。目的二是确定生殖细胞的致病性 通过评估体外受精后的后代结局和精子来源的ncRNA进行的表观遗传变化 注射目标3将确定父亲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外谱系，使用单细胞转录组学分析， 单细胞水平的囊胚和来自对照、HFD和 HFD+ CANA治疗的父亲。目的4将检验AMPK-Tet 2通路（葡萄糖的介导物）的假设。 介导体细胞中的表观遗传调节，也介导生殖细胞中葡萄糖诱导的变化 表观基因组我们将分析AMPK在体内激活对野生型或Tet 2-null精子中5 hmC的影响。 小鼠和后代表型。总之，确定因果机制，以回应父亲的 高血糖和肥胖-及其逆转-将提供关键的新信息，以指导新的 以家长为重点的可翻译方法，以减少后代的疾病负担。

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.