Regulation of the Human Ghrelin Promoter Activity by Transcription Factors, NF-κB and Nkx2.2.

To examine the gene expression of ghrelin, a growth hormone releasing and appetite stimulating hormone from stomach, we constructed human ghrelin promoter-reporter vectors and analyzed the promoter activity. The ghrelin promoter activity was high when cultured cells that express ghrelin mRNA endogenously like TT or ECC10 cells were used, indicating that these cells contain factors necessary for full expression of the human ghrelin gene. The human ghrelin promoter contains both positive and negative regulatory regions. A transient decrease of the promoter activity was found when the reporter vector with the -1600 fragment of the human ghrelin promoter was transfected into cultured cells. We then examined the effect of several transcription factors on the ghrelin promoter activity and found that NF-κB suppressed and that Nkx2.2, a homeodomain-containing transcription factor that is important for ghrelin cell development in pancreas, activates the promoter activity. These transcription factors may be possible targets for the control of ghrelin gene expression.

Enzymatic characterization of GOAT, ghrelin O-acyltransferase.

Ghrelin is a gastric peptide hormone in which serine 3 (threonine 3 in frogs) is modified primarily by an n-octanoic acid; this modification is essential for ghrelin's activity. The enzyme that transfers n-octanoic acid to the third serine residue of ghrelin peptide has been identified and named GOAT for ghrelin O-acyltransferase. GOAT is the only known enzyme that catalyzes the acyl modification of ghrelin and specifically modifies the third amino acid serine and does not modify other serine residues in ghrelin peptides. GOAT prefers n-hexanoyl-CoA over n-octanoyl-CoA as the acyl donor, although in the stomach n-octanoyl form is the main acyl-modified ghrelin and the concentration of n-hexanoyl form is very low. Moreover, a four-amino acid peptide derived from the N-terminal sequence of ghrelin can be modified by GOAT, indicating that these four amino acids constitute the core motif for substrate recognition by the enzyme.

Structure, regulation and function of ghrelin.

Ghrelin is a stomach hormone that acts as an endogenous ligand of orphan G-protein-coupled receptor. Ghrelin is a 28-amino acid peptide existing in two major forms: n-octanoyl-modified ghrelin, which possesses an n-octanoyl modification on serine-3 and des-acyl ghrelin. Fatty acid modification of ghrelin is essential for ghrelin-induced growth hormone release from the pituitary and appetite stimulation. This acyl-modification of ghrelin is catalysed by ghrelin-O-acyl transferase recently identified. Despite the number of innovative advancements in this field of research, there are still many aspects of ghrelin function and biosynthesis process that remain to be clarified. Here, we review the current understanding of the structure, regulation and function of ghrelin; this review is intended for researchers who will be involved in this field in the future.

Ghrelin O-acyltransferase (GOAT) has a preference for n-hexanoyl-CoA over n-octanoyl-CoA as an acyl donor.

Ghrelin is a peptide hormone in which serine 3 is modified by n-octanoic acid through GOAT (ghrelin O-acyltransferase). However, the enzymological properties of GOAT remain to be elucidated. We analyzed the in vitro activity of GOAT using the recombinant enzyme. Unexpectedly, although the main active form of ghrelin is modified by n-octanoic acid, GOAT had a strong preference for n-hexanoyl-CoA over n-octanoyl-CoA as an acyl donor. Moreover, a four-amino acid peptide derived from the N-terminal sequence of ghrelin can be modified by GOAT, indicating that these four amino acids constitute the core motif for substrate recognition by the enzyme.

The role of ghrelin and ghrelin-receptor gene variants and promoter activity in type 2 diabetes.

BACKGROUND: Ghrelin and its receptor play an important role in glucose metabolism and energy homeostasis, and therefore they are functional candidates for genes carrying susceptibility alleles for type 2 diabetes. METHODS: We assessed common genetic variation of the ghrelin (GHRL; five single nucleotide polymorphisms (SNP)) and the ghrelin-receptor (GHSR) genes (four SNPs) in 610 Caucasian patients with type 2 diabetes and 820 controls. In addition, promoter reporter assays were conducted to model the regulatory regions of both genes. RESULTS: Neither GHRL nor GHSR gene SNPs were associated with type 2 diabetes. One of the ghrelin haplotypes showed a marginal protective role in type 2 diabetes. We observed profound differences in the regulation of the GHRL gene according to promoter sequence variants. There are three different GHRL promoter haplotypes represented in the studied cohort causing up to 45% difference in the level of gene expression, while the promoter region of GHSR gene is primarily represented by a single haplotype. CONCLUSION: The GHRL and GHSR gene variants are not associated with type 2 diabetes, although GHRL promoter variants have significantly different activities.

The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system.

Immunohistochemical analysis revealed the presence of a gut-brain peptide, neuromedin U (NMU), in the suprachiasmatic nucleus (SCN), which is the site of the master circadian oscillator. The expression of NMU mRNA exhibited a circadian rhythm, with the peak expression in the SCN occurring at CT4-8h. The two NMU-binding receptors (NMU-R1 and NMU-R2) were also expressed in the SCN, but their phase angles were different. Intracerebroventricular injection (ICV) of NMU induced the expression of Fos protein in the SCN cells and caused a phase-dependent phase shift of the circadian locomotor activity rhythm. The magnitude of the phase shift was dose dependent. This NMU-induced phase shift was of the nonphotic type. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed increases in the expression in the SCN of immediate early genes, such as c-fos, NGFI-A, NGFI-B, and JunB. Furthermore, ICV injection of NMU increased the expression of Per1, but not Per2, in the SCN. These results indicate that NMU may play some important role in the circadian oscillator by exerting an autocrine or paracrine action in the SCN.