Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress.
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-I, 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.
Kineman
Rhonda D
RD
Section of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA.
Gahete
Manuel D
MD
Luque
Raul M
RM
D000818:Animals; D001483:Base Sequence; D005215:Fasting / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D054439:Ghrelin; D007031:Hypothalamus / Q000378:metabolism*; D007438:Introns; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008820:Mice, Obese; D008969:Molecular Sequence Data; D009765:Obesity / Q000378:metabolism; D036361:Peptide Hormones / Q000235:genetics* / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013312:Stress, Physiological / Q000378:metabolism*
D054439:Ghrelin; D036361:Peptide Hormones; D020033:Protein Isoforms; D012333:RNA, Messenger