ETAPAS DEL FUNCIONAMIENTO DEL SISTEMA DE CRÉDITO

I used a full length pre-pro-Ghrelin 28 riboprobe to analyse the mouse Ghrelin expression pattern in several tissues by in situ hybridisation. In stomach I found the same pattern in mouse as reported in the rat with very strong Ghrelin mRNA expression from the neck to the base of the oxyntic gland (Figure 8.2). Dipping the sections in photographic emulsion showed that mouse Ghrelin was expressed heavily in cells at the base of the microvilli near the nerve plexus (Figure 8.3). Analysis of gut sections showed that gut Ghrelin was restricted to few scattered, but highly labelled cells (Figure 8.2 and 8.3). Stomach and gut Ghrelin in situ hybridisations were left to develop for two days, while other tissues only showed signals after two weeks of exposure to film. Ghrelin expression in mouse testis appeared restricted to some tubules, but not others (Figure 8.2). Ghrelin expression in the heart was not visible on film, but analysis at higher resolution showed only two Ghrelin positive cells in 6 heart sections (data not shown). Histology of these frozen sections was not ideal and the exact localisation or nature of these cells was unclear. Analysis of Ghrelin expression in the brain showed that mRNA expression levels were extremely low, if present at all. The anti-sense hybridisation signal was hardly distinguishable above a sense signal (Figure 8.2). Higher resolution analysis showed no specific labelling in the hypothalamus and only few scattered cells seemed to be labelled, but had no distinct localisation pattern in the brain. No Ghrelin expression was found in the kidney, thyroid, lung or pituitary with this technique.

Chapter 8; Ghrelin - a novel growth hormone secretagogue A nti-sense S e n s e sto m a c h gut te s tis brain

Figure 8.2 Ghrelin expression in mouse tissues

12pm frozen tissue sections were hybridised with a full length Ghrelin anti-sense (left panel) and sense (right panel) riboprobe. Stomach and gut were exposed for two days, while testis and brain needed two weeks exposure. Strong Ghrelin expression was apparent in the stomach and specific cells in the gut. Some tubules in the testis showed Ghrelin hybridisation as well, while in the brain no distinct hybridisation signal above background was visible. Scale bar =1 mm

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Figure 8.3 High resolution imaging of mouse Ghrelin expression in stomach and gut

Ghrelin in situ hybridisation tissue sections were dipped into photographic emulsion, developed and counterstained with cresyl fast-violet. In the stomach Ghrelin was expressed strongly in specific cells at the base of microvilli near the nerve plexus. In the gut Ghrelin expression was distributed sparsely, but expression in single cells was high.

Chapter 8: Ghrelin - a novel growth hormone secretagogue

Kojima et al. have identified an acromegalic patient suffering from Ghrelin secreting carcinoids (personal communication I.C.A.F. Robinson), thus indicating that overproduction of gastric Ghrelin will have an effect on the GH axis. Since in other acromegalic patients ectopic GHRH over-expressing tumours cause GH hypersecretion, and GHRH has previously been identified in the GI tract (Bosman et a l, 1984), I aimed to analyse whether GHRH was expressed in the mouse stomach and intestine and if so, if it might co-localise with Ghrelin. In situ hybridisation and subsequent high resolution imaging using a GHRH mRNA riboprobe did not detect any GHRH mRNA expression in the stomach or gut of WT mice.

In order to compare Ghrelin expression in rGHRH-hGHS-R and WT mice an RNAse protection assay measuring both Ghrelin 28 and Ghrelin 27 was established. Ghrelin 28 was used as a probe, such that hybridisation of the Ghrelin 28 probe to Ghrelin 27 would result in a base mismatch and be digested by the RNAse into a large 307 bp fragment and a smaller 115bp fragment. The assay was optimised for fragments above 150bp, so I chose to focus on the larger band for Ghrelin 27. High levels of Ghrelin expression were seen in the stomach, with no difference in stomach Ghrelin expression in rGHRH- hGHSR transgenic and WT mice (Figure 8.4). The relative expression levels of Ghrelin 27:28 in the stomach were 1:1.7 (n=4). Of the other tissues only Ghrelin expression in the gut was detectable. The levels of hypothalamic Ghrelin expression were so low that they are undetectable by this very sensitive method. However, by RT-PCR using mouse Ghrelin specific primers for exon 2 and 3 on the same samples, Ghrelin expression could be found in all tissues including hypothalamus. In both assays, RFA and RT-PCR, RNA amounts were controlled by measuring p-actin levels in the same samples. These data indicate that the amount of Ghrelin RNA in other tissues than stomach and gut is extremely low. Its physiological significance in all these tissues is thus not clear, especially since the type la GHS-R is not expressed in peripheral tissues (Howard et al,

Ghrelin 28 Ghrelin 27 RNAse Protection: 1 2 3 4 5 6 7 8 9 RT-PCR: 1 stomach WT 2 stomach rGHRH-hGHS-R 3 hypothalamus WT 4 hypothalamus rGHRH-hGHS-R 5 heart WT 6 testis WT 7 gut WT 8 kidney WT 9 brain WT

Figure 8.4 Ghrelin RNAse protection assay and RT-PCR

For RNAse protection assay (RPA) 10|ig of RNA were loaded per tissue and hybridised with a Ghrelin RNA probe over night. RNAse digestion and subsequent gel-separation revealed that Ghrelin 27 and 28 were measurable only in WT and rGHRH-hGHS-R stomach and WT gut. RT-PCR with mouse Ghrelin specific primers on 0.5 pg cDNA showed expression in all tissues analysed.