Endocrine Abstracts

Background: We have recently demonstrated universal and high levels of expression of growth hormone in the human colon, despite the absence of GHRH or its receptor. Although ghrelin, recently shown to be an additional endogenous GH secretagogue, is expressed in the human colon, little is known about its distribution within the colon or function and whether any effects are GH-dependent.

Aims: To investigate the mRNA expression of ghrelin and its receptor (GHS-R) in the normal colon and investigate its in vitro effects on colonic epithelial cell proliferation.

Methods: RNA was extracted from 15 paired samples of sigmoid and caecal colonic tissue obtained during routine colonoscopy. mRNA levels were quantified using a real time RT-PCR assay and expressed as copy number/µg total RNA. HT-29 cells were grown in 96 well plates in serum free media for 72 hours with or without ghrelin (10^-9-10^-6M). Proliferation was assessed by the MTS assay at 24, 48 and 72 hrs.

Results: All samples expressed ghrelin mRNA (median 1.1x10⁵, range 4x10²-5.4x10⁵ copies/µg total RNA), whereas GHS-R mRNA could not be detected in any of the biopsies. There was no difference in ghrelin mRNA levels between the sigmoid colon and the caecum. Interestingly, there was a significant correlation between ghrelin and c-myc mRNA in the caecum (R=0.47; p<0.05) indicating a possible stimulatory effect on this proto-oncogene. There was also a significant correlation with IGF-IR (R=0.77; p<0.00005), IGFBP-3 (R=0.72; p<0.0005) and Cox-2 (R=0.52; p<0.01), but no correlation with GH or GH-R mRNA. When ghrelin was added to HT-29 cells at a concentration of 10^-9M, increased proliferation was observed after 24h on 3 consecutive experiments (mean increase 33%, range 27-36%; p<0.02-0.0001). Higher concentrations (10^-7 and 10^-6M) resulted in a significant decrease in cell number at 48 and 72 hours.

Conclusions: Our data provide clear evidence for an effect of ghrelin on cell growth and suggest that this regulation involves the c-myc proto-oncogene and Cox-2. These effects of ghrelin are not exerted through the GHS-R, but do appear to be associated with IGF-IR and IGFBP-3 expression.