MODELO DE SISTEMAS VIABLES (MSV)

(B ennett et al., 1998). To analyse, whether orally administered L-163,255 treatment altered ARC NPY expression the brains from all rats were cut into 12|im sections on a cryostat and their ARC NPY mRNA expression was analysed by in situ hybridisation with a rat NPY riboprobe. No significant changes in NPY mRNA expression were measured, possibly due to high background variability in all groups (Figure 9.3). Interestingly though, mean NPY mRNA was higher in all L-163,255 treated groups compared to their untreated control animals, except in the male treated Tgr rats.

If this study was repeated, a grain count of NPY mRNA expression per neurone might be a more concise method than densitometry.

5000- 4000- 3000- (0 c <D ■o

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Figure 9.3 ARC NPY mRNA expression in orally L-163,255 treated Tgr and WT rats

Hypothalamic ARC sections were used for an in situ hybridisation with a rat NPY riboprobe and the autorads were analysed by densitometry. Although in most L-163, 255 treated groups the NPY mRNA content seemed higher, no significant changes were found between any of the groups (n=8, n.s., Mann-Whitney Test).

9.1.3 Discussion

Previously L-163,255 had been given to pigs through oral gavage and in feed and was shown to release GH from the pituitary at doses between 30 and 300p.g/kg (Chang et ai,

AUC measurements over the initial 12-hour period and increased mean IGF-I levels over the treatment period. In addition, Tgr rats had previously been shown to respond to pulsatile s.c. GHRP-6 treatment with enhanced body weight gain. Therefore, I used Tgr rats to test the ability of the orally active MK-0677 analogue L-163,255 to promote growth. A dose of ~13mg/kg was given ad libitum to rats in their drinking water.

Female treated Tgr rats showed significantly enhanced body weight gain in response to the L-163,255 treatment compared to Tgr untreated controls. Comparison of their body weight gain with treated WT rats showed that the treated Tgr weight gain was always higher, but this was only significant on day 8 of the study. Neither male WT or Tgr, nor female WT rats responded to the L-163,255 treatment with enhanced body weight gain. No significant difference was shown between male and female treated Tgr pituitary GH contents compared to their untreated controls. However, females showed a higher mean GH content than their controls, which does not fully explain, but might add to their enhanced body weight gain in response to GHS treatment. Similarly, male treated Tgr rats were the only GHS treated group that did not show slightly elevated mean NPY mRNA expression.

Essentially, it remains unclear why female Tgrs responded to the treatment and male Tgrs did not. One explanation might lie in the differences in GH secretion and GH responses of Tgrs to acute i.v. injections of GHRP-6. While male Tgrs are impaired in GH secretion pattern and GH responses to GHRP-6 are attenuated compared to WT controls, female Tgr’s GH secretion and GH responses are normal. This might explain, why female Tgr rats responded better to the L-163,255 treatment than male animals. Also, data from the L-163,255 study were consistent with reports showing that GH responses to GHSs are greater and more consistent in female rats (Bercu et a l, 1991; Sartor et a l, 1985). Many studies with GHS treatment have therefore used female animals, e.g. (Bercu et a l, 1992; Carmignac e ta l, 1998; McDowell e ta l , 1995; Svensson et a l, 2000; Thomas et a l, 2000). Furthermore in some cases E2 was shown to increase GH responses to GHRP-6 (Mallo et a l, 1993) and Carmignac et a l pointed out that E2 increased GHS-R transcription (Carmignac et a l, 1998).

These data gave a possible explanation as to why female Tgrs responded better than male Tgr, but then why didn’t WT females grow? Growth stimulation is best demonstrated in GH deplete animals and GH deficient Tgr rats have so far shown the

Chapter 9: GHS treatment studies in transgenic m ice and rats

most impressive body weight gain in response to GHSs, compared to other GHS treatment studies in normal rats (Wells et a l, 1997). Dwarf rats {dw/dw) have previously been shown to have elevated GHS-R expression levels (Bennett et a l, 1997) and if it holds true that GH deficiency in Tgr rats also causes enhanced hypothalamic GHS-R levels, their enhanced responsiveness might also be explained by enhanced hypothalamic GHS-R levels. Thus measurement of GHS-R mRNA levels in the animals of this study would have been helpful.

As mentioned earlier, GHS treatment can affect body weight gain as enhanced feeding and fat mass rather than growth (Kuriyama et a l, 2000; Locke et a l, 1995; Okada et a l,

1996). Studies giving L-163,255 i.c.v. showed that food intake was not affected in rats (personal communication D.F.Carmignac), suggesting that the body weight gain observed here in female Tgrs might indeed be growth. This cannot be stated with certainty, since in this study no measurement of food intake, fat mass or tibia length/growth plate width were performed and the slightly enhanced ARC NPY levels might suggest an effect on the metabolic axis. At the moment it is thus unclear, whether L-163,255 affected the GH or metabolic axis in female Tgrs. Especially in view of the recent study by Tschop et a l, which showed that Ghrelin, an endogenous ligand for the GHS-R, given i.c.v enhanced food intake and caused increased body weight gain as a result of reduced fat utilisation (Tschop et a l, 2000), more studies to investigate the growth/metabolic effect of oral L-163,255 treatment are needed.

Nevertheless, data from this study showed that female, GH deficient animals were the most promising animals to elicit growth responses to GHS treatment. I suspected that this might be due to a contribution of elevated GHS-R expression levels and sensitivity to GH correction.

9.2

GHS treatment of rGHRH-hGHS-R and WT mice

9.2.1 Introduction

The above described L-163,255 study suggested that enhanced GHS-R mRNA expression might contribute to enhanced responsiveness to GHS treatment. Our previous experiments suggested that transgenic mice over-expressing the GHS-R in the hypothalamus might show increased responsiveness to acute i.v. injections of GHRP-6 in vivo. I therefore aimed to investigate, whether enhanced hypothalamic GHS-R

expression in GHRH neurones was able to enhance growth responses to long-term GHS treatment in rGHRH-hGHS-R transgenic mice.

Previously McDowell et a l had described experiments in which female rats were treated either continuously or intermittently s.c. with a GHS for 14 days, resulting in an enhanced body weight gain compared to saline treated rats, which was much greater with twice daily s.c. injections than with continuous s.c. exposure (McDowell et a l,

1995). Similarly, Thomas et a l confirmed that continuous s.c. infusion of GHRP-6 had an initial effect on weight gain in rats but failed to produce a sustained increase in weight gain altogether (Thomas et a l, 2000). Fairhall et al. had shown that GH responses to GHSs were dependent on the pattern of GHS administration with more consistent GH responses when GHSs were administered with less frequent i.v. injections rather than continuous GHS infusion (Fairhall e ta l, 1995). Moderate body weight gain responses to GHSs have also been achieved in rats by once daily oral or s.c. treatment (Bowers et a l, 1984; Hansen et a l, 1999), pulsatile i.v. infusion (Wells et a l, 2000; Wells et a l, 1997) and continuous s.c. infusion (Svensson et a l, 2000).

Except for the Svensson et a l study (Svensson et a l, 2000) continuous infusion had shown desensitisation to GHS (McDowell e ta l, 1995; Thomas e ta l, 2000). Since in my study with oral L-162,255 administration it had proven difficult to control the exact dosage per animal per day, a once daily i.p. injection in mice was chosen instead. Furthermore, twice daily s.c. GHS injections had proven effective in rats (McDowell et a l, 1995) and I aimed to investigate the effectiveness of this injection schedule to promote body weight gain in rGHRH-hGHS-R transgenic mice. Transgenic rGHRH- hGHS-R mice and their WT littermates were thus exposed to GHRP-6 injections over 9 -14 days and the effects on body weight analysed.

9.2.2 GHRP-6 (25fig/day Ip.) treatment fo r 14 days in female rGHRH-hGHS-R transgenic mice

Groups of age-matched 11-13 week old female transgenic rGHRH-hGHS-R and WT mice were injected i.p. daily with or without 25|ig GHRP-6 in 200)l i1saline (WT saline: n=5, WT GHRP-6: n=4, T saline: n=5, T GHRP-6: n=6). The body weight of these animals was recorded every three days. After 14 days treatment, mice were sacrificed and their ovarian fat pads and the soleus muscle weight measured. Brain and pituitary