Physiological Factors Driving Maturation of Neonatal Beta Cells

描述(申请人提供)：这项建议侧重于了解出生后第一个月发生的主要生理变化如何调节关键表型�细胞基因表达的急剧变化，并推动�细胞的功能成熟。令人惊讶的是，人们对�细胞成熟的后期知之甚少。这一点具有实际意义，因为在体外，人类胚胎干细胞-胰岛素产生细胞缺乏葡萄糖反应性胰岛素分泌，这是功能正常的�细胞的特征。新生的啮齿动物胰岛也缺乏这种葡萄糖反应性，因此可以作为诱导成熟过程的模型。在出生后第一个月，随着葡萄糖刺激的胰岛素分泌逐渐发展，我们发现胰岛基因表达的多种模式，大多数在P7至9天之间有明显的拐点，在P15左右再次出现。虽然我们已经证明，腺病毒介导的MafA诱导的胰岛素分泌(GSIS)几乎达到成人水平，但MafA和其他必要基因的发育增强必须受到生理刺激的调节。与成人非常不同的动态新生儿环境提供了这些变化的图景：在P1，血糖水平低，甲状腺激素(T3和T4)水平低，皮质酮(大鼠的主要糖皮质激素)和催乳素水平低，饮食只有牛奶。血清T4水平在P7左右开始变化，在P13左右达到高峰，而瘦素也在P8左右升高；皮质酮和催乳素在P15左右出现激增。这些激素水平的上升时间表明，它们是新生儿�细胞功能所需的生理调节因子。为了解决这个问题，我们有三个具体目标。具体目的1.探讨甲状腺激素促进�细胞成熟的机制。我们的初步数据显示，T3诱导葡萄糖反应性胰岛素分泌，这是由转录因子MafA介导的。我们建议通过实验来剖析T3‘S对MafA和其他基因诱导功能成熟的调控机制。具体目的2.确定候选因子T3、瘦素、皮质酮和催乳素对�细胞成熟的影响。新生儿的环境与成人有很大的不同，而且是动态的。我们假设，不同的生理因素调节�细胞的成熟，并特别受到三种激素的吸引，除了甲状腺激素，这可能起关键作用：血浆瘦素浓度在P7拐点附近激增，而皮质酮和催乳素浓度逐渐增加，直到P15左右，第二个关键拐点。具体目的3.确定影响新生大鼠B细胞成熟的生理因素是否同样指导未成熟的人胰腺祖细胞在体外的功能成熟。在这个翻译目标中，我们的目标是将前两个目标中获得的体内成熟的生理信号的知识转移到胎儿胰腺细胞中。

DESCRIPTION (provided by applicant): This proposal focuses on understanding how the major physiological changes occurring during the first postnatal month regulate the sharp changes in expression of key phenotypic � cell genes and drive functional maturation of � cells. Surprisingly little is known about the later stages of � cell maturation. This has assumed practical importance because in vitro human ES-derived cells insulin-producing cells lack the glucose-responsive insulin secretion that is characteristic of a functional � cell. Neonatal rodent islets also lack this glucose responsiveness and so serve as a model for the induction of the maturation process. Across the first postnatal month as the glucose-stimulated insulin secretion gradually develops, we found multiple patterns of islet gene expression, most with striking inflection points between P7 and 9 days and again about P15. While we have shown that adenoviral-mediated expression of MafA induced glucose- stimulated insulin secretion (GSIS) almost to adult levels, the developmental enhancement of MafA and other necessary genes must be regulated by physiological stimuli. The dynamic neonatal environment that is very different from that of the adult provides the landscape of these changes: at P1 blood glucose levels are low, as are levels of thyroid hormones (T3 and T4), of corticosterone (the main glucocorticoid of the rat) and of prolactin, and the diet is only milk. Serum T4 levels start changing around P7 and peak at P13 while leptin surges also about P8; there are surges of corticosterone and prolactin around P15 The timing of these hormonal surges suggests that they are the physiological regulators needed for neonatal � cells to become functional. To address this question we have three specific aims. Specific aim 1. To determine the mechanisms for thyroid hormone enhancement of � cell maturation. We show preliminary data that T3 induces glucose responsive insulin secretion and that is mediated by the transcription factor MafA. We propose experiments to dissect the mechanism of T3's regulation of MafA and other genes induced for functional maturation. Specific aim 2. To determine the importance of the candidate factors T3, leptin, corticosterone and prolactin on � cell maturation. The environment of the neonate is very different than that of the adult and dynamic. We hypothesize that various physiological factors regulate the maturation of the � cell and are particularly attracted to three hormonal candidates, in addition to thyroid hormone, that may play key roles: plasma leptin concentrations surge at about the P7 inflection point, while corticosterone and prolactin concentrations are gradually increasing until a surge at about P15, the second key inflection point. Specific aim 3. To determine if the physiological factors that are effective in maturing the neonatal rat B cell will similarly direct in vitro functional maturation of the immature human pancreatic progenitors. In this translational aim our goal is to transfer to fetal human pancreatic cells the knowledge of the physiological signals for in vivo maturation gained in the first two aims.