Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress
Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress
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DOI：
10.1677/jme-06-0026
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发表时间：
2007-05-01
影响因子：
3.5
通讯作者：
Luque, Raul M.
中科院分区：
文献类型：
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作者：
Kineman, Rhonda D.;Gahete, Manuel D.;Luque, Raul M.
小鼠ghrelin基因含有5个外显子（Ex），其中Ex2-Ex5编码117个氨基酸的前原蛋白，其被加工以产生28个氨基酸的成熟肽。目前的研究检查了垂体（PIT）和下丘脑（HPT）ghrelin表达是否在响应禁食时上调，在肥胖时下调，如先前在胃中报道的那样。在建立实时定量RT-PCR系统以准确评估PIT和HPT ghrelin mRNA水平变化的过程中，我们观察到位于Ex2和Ex3中的引物组扩增了包含整个内含子2（In2）的ghrelin转录物。使用位于整个生长素释放肽基因的多个引物组的RT-PCR产物的大小和序列分析表明，In2-生长素释放肽变体含有Ex2和Ex3，但缺乏Ex1、Ex4和Ex5。在胃提取物中未检测到In2-ghrelin变体mRNA，而在PIT和HPT中的表达水平分别是天然ghrelin转录物的10倍和50倍。In2-ghrelin变体mRNA水平在禁食24小时后的PIT中增加，而在饮食诱导的肥胖小鼠的HPT和PIT中降低。这些变化可能是由于循环胰岛素或IGF-1的变化，因为在小鼠HPT细胞系（N6）和原代小鼠PIT细胞培养物中In2-ghrelin变体表达均降低。In2-ghrelin变体mRNA水平依赖于PIT和HPT中的能量摄入的事实表明，该转录本可能编码在协调对代谢应激的神经内分泌反应中重要的肽。
The mouse ghrelin gene contains 5 exons (Ex), with Ex2-Ex5 encoding a 117 amino acid preproprotein that is processed to yield a 28 amino acid mature peptide. The current study examined if pituitary (PIT) and hypothalamus (HPT) ghrelin expression is up-regulated in response to fasting and down-regulated in obesity, as previously reported in the stomach. In the process of establishing a quantitative real-time RT-PCR system to accurately assess the changes in PIT and HPT ghrelin mRNA levels, we observed that primer sets located in Ex2 and Ex3 amplified a ghrelin transcript that contained the entire intron 2 (In2). Size and sequence analysis of RT-PCR products using multiple primer sets located throughout the ghrelin gene suggested that the In2-ghrelin variant contains Ex2 and Ex3, but lacks Ex1, Ex4, and Ex5. In2-ghrelin variant mRNA was not detected in stomach extracts, while expression levels were 10- and 50-fold greater than that of the native ghrelin transcript in the PIT and HPT respectively. In2-ghrelin variant mRNA levels increased in the PIT after 24 h fasting and decreased in the HPT and PIT of diet-induced obese mice. These changes may be due to the changes in circulating insulin or IGF-1, since both decreased In2-ghrelin variant expression in a mouse HPT cell line (N6) and in primary mouse PIT cell cultures. The fact that In2-ghrelin variant mRNA levels are dependent on energy intake in the PIT and HPT suggests that this transcript may encode a peptide important in coordinating the neuroendocrine response to metabolic stress.