Identification of a mouse ghrelin gene transcript that contains intron 2 and is regulated in the pituitary and hypothalamus in response to metabolic stress

Abstract

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.

Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer

“…7A, treatment with 10 nM insulin significantly increased the expression of in2c-ghrelin in the LNCaP (1.3-fold, P!0.05) and 22Rv1 (4.4-fold, P!0.05) prostate cancer cell lines. Interestingly, insulin also significantly upregulated the expression of a recently reported intron 1-retained transcript, in1-ghrelin (Kineman et al 2007, Gahete et al 2010. A 2.2-fold increase in expression was observed in LNCaP cells and a 2.8-fold increase in 22Rv1 cells (Fig.…”

“…We demonstrate that this novel transcript, as well as the recently described in1-ghrelin transcript (Kineman et al 2007, Gahete et al 2010, is upregulated in response to insulin treatment in the 22Rv1 and lymph node carcinoma of the prostate (LNCaP) prostate cancer cell lines and, therefore, they may have a role in prostate cancer progression, particularly in cases of hyperinsulinaemia.…”

“…Indeed, the ghrelin peptide has been shown to play an immunoregulatory role (Baatar et al 2011). N IA IA IA IA IA IB IB IC IIB IIB IIIB IIIC IIIC IIIC IIIC IV IIIA IIIA III III IIB I I I IIA IIA IIA IIA IIA IIA IIA IIA IIB IIB IIB IIIA IIIA IIIA IIIA IIIA IIIA IIIC IIIC IIIC N N N N N N N N N N II II II II II II II II II II II III III III III III III III IV I I I I I I I I I I I IA IA IB IB IB IB IB A number of other ghrelin isoforms have also been described previously (Seim et al 2009(Seim et al , 2011a, including exon 3-deleted preproghrelin ) and the intron 1-retained transcript (originally termed in2-ghrelin but renamed in1-ghrelin using the current ghrelin gene nomenclature; Kineman et al 2007, Gahete et al 2010. In1-ghrelin would encode a 117 amino acid polypeptide that includes the signal peptide of preproghrelin, the first 12 amino acids of the 28 amino acid ghrelin peptide sequence and a novel 81 amino acid C-terminal peptide, but not obestatin (Gahete et al 2011).…”

“…In1-ghrelin would encode a 117 amino acid polypeptide that includes the signal peptide of preproghrelin, the first 12 amino acids of the 28 amino acid ghrelin peptide sequence and a novel 81 amino acid C-terminal peptide, but not obestatin (Gahete et al 2011). In1-ghrelin is regulated in response to metabolic stress in mice (Kineman et al 2007) and is down-regulated in particular brain regions in Alzheimer's disease in humans (Gahete et al 2010) and upregulated in breast cancer (Gahete et al 2011).…”

“…Insulin treatment also up-regulates ghrelin expression in mouse stomach oxyntic glands (which are the major site of ghrelin expression; Toshinai et al 2001) and in adipocytes (Gurriaran-Rodriguez et al 2011). Conversely, insulin down-regulates the expression of wild-type and in1-ghrelin mRNA in the mouse hypothalamus, where it has an effect on appetite regulation (Kineman et al 2007, Fick et al 2009). The regulation of ghrelin in cancer has yet to be established, and this is the first study to demonstrate regulation of ghrelin transcripts by insulin in cancer.…”