Increased expression of the mRNA encoding the somatostatin receptor subtype five in human colorectal adenocarcinoma.

Numerous studies have suggested that the antiproliferative potency of somatostatin (SS) analogues may be an efficient tool to improve the prognosis of colorectal cancer. In order to facilitate current efforts to design potent antitumour SS analogues, we studied the distribution of human SS receptors (hsst1-5) mRNAs in a large set of tumoural and normal colonic tissues. Localisation of hsst1-5 mRNAs in normal and tumoural tissues was performed by in situ hybridisation using radioactive antisense or sense riboprobes. Semi-quantitative analysis of hsst5 mRNA was performed using a computerised image analysis system. Hsst binding sites were characterised by studying the relative potency of SS14, SS28 or SS analogues in displacing [(125)I]Tyr degrees -d-Trp(8)-SS14 bound to HT29-D4 cells. Hsst5 mRNA was by far the most expressed subtype in both normal and transformed epithelial cells as well as in the HT29-D4 cell line. An increased expression of hsst5 mRNA was found in tumours. Hsst1 mRNA was expressed preferentially as clusters in immune cells in lamina propria and in stroma close to the tumour. A low expression of hsst4, hsst3 and hsst2 was seen in normal and tumoural tissue. In HT29-D4, binding experiments with SS14 demonstrated the existence of one SS binding class (K(d)=524 nM, B(max)=1fmol/10(6 )cells). In competition binding studies, SS28 and BIM23268 (an analogue that shows preferential specificity towards hsst5) effectively inhibited binding of [(125)I]Tyr degrees -d-Trp(8)-SS14 (IC(50)=15 and 157 nM respectively), while BIM23197 (an analogue that shows preferential affinity for hsst2) was ineffective. Our results show a high expression of hsst5 mRNA in human tumoural colonic tissue, while hsst5 protein is the predominant hsst protein subtype in a tumoural colonic cell line.

IGF-I/IGFBPs system response to endotoxin challenge in sheep.

Endotoxin (LPS), a membrane component of gram-negative bacteria produces multiple endocrine and metabolic effects that mimic those seen in acute sepsis. It induces species-dependent alterations of the growth hormone (GH) axis that may participate in the shift of the metabolism towards catabolic events. Humans and sheep show increased GH secretion in response to LPS, as opposed to rats, which have been the most studied. The purpose of our work was to evaluate the effects in intact rams of an acute intravenous administration of a high dose of LPS on the insulin-like growth factor (IGF)-I/IGF-binding proteins (IGFBPs) system and to analyse the temporal relationship of GH axis changes with those of several hormonal and metabolic parameters such as somatostatin, cortisol, insulin, and glucose. LPS induced a late moderate decrease of total IGF-I plasma levels following a 5-h steady-state period (-26.6+/-4. 2%, P<0.05, 9 h after LPS), despite a biphasic and sustained increase of GH secretion in the same animals (2.48+/-0.39 ng/ml 2 h after LPS and 2.7+/-0.37 ng/ml 5 h after LPS vs 0.77+/-0.10 before LPS; Briard et al. 1998a). Western ligand blot analysis in IGFBPs showed an early short-lasting increase in IGFBP-1 (188.8+/-39% P<0. 05, 3 h after LPS). No significant change was seen for either IGFBP-2, -3 or -4. We observed a marked and sustained increase in cortisol (128.18+/-7.21 ng/ml 3 h after LPS, vs 21.17+/-4.22 before LPS). Insulin also increased (27.69+/-3.90 microU/ml 3 h after LPS, vs 13.48+/-1.69 before LPS) and its burst coincided with that of IGFBP-1. Moderately decreased IGF-I and increased IGFBP-1 plasma levels contrasted with the sustained increase in GH secretion that we recently described, thereby suggesting that endotoxin causes a state of resistance to GH. This may be exacerbated by reduced IGF-I bioavailability and/or action, and which may participate in the pathophysiology of the catabolic state seen in sepsis. The temporal analysis of hormone responses suggests that endotoxin-induced alterations of the IGF-I/IGFBPs system may involve the prolonged and substantial somatostatin rise that we recently demonstrated, together with an increase in glucocorticoid and cytokine as more generally assumed.

No loss of sst receptors gene expression in advanced stages of colorectal cancer.

As demonstrated by several studies, the pan-inhibitory peptide somatostatin (SS) is implicated in a large variety of physiological processes in the gastrointestinal tractus. SS inhibits hormonal and gastric acid secretions, and decreases gastric and intestinal motility, mesenteric blood flow and intestinal absorption. In vitro and in vivo studies showed also that the antiproliferative potency of SS analogs may be a target to improve the prognosis of colorectal cancer. Here we report the expression profile of the five SS receptor subtypes (hsst1-5) mRNAs in a large set of tumoral and normal colon. Using reverse transcription-PCR, we showed that hsst5, hsst1 and hsst2 mRNA subtypes were the most frequently expressed hsst mRNA subtypes in normal and pathological colon. Interestingly, we found that the frequency of hsst5 mRNA expression in the left colon was significantly higher in tumors than in normal samples: 81. 2% (13/16) and 36.4% (4/11) respectively (0.025>P>0.01, chi2 test with Yates' correction). We did not find any influence of Dukes' stage on hsst mRNAs expression. Of interest, no loss of hsst2 and hsst5 mRNA expression in advanced stages was noted. Some differences in the frequency of expression of hsst mRNAs according to the origin of the tissue (left or right colon) were evident. The expression of hsst5 and hsst2 mRNA in advanced colorectal carcinoma associated with the development of new SS analogs boost the relevance of colorectal cancer treatment by somatostatin analogs.

Hypothalamic mediated action of free fatty acid on growth hormone secretion in sheep.

Experimental data suggest that elevated FFA levels play a leading role in the impaired GH secretion in obesity and may therefore contribute to the maintenance of overweight. GH has a direct lipolytic effect on adipose tissue; in turn, FFA elevation markedly reduces GH secretion. This suggests the existence of a classical endocrine feedback loop between FFA and GH secretion. However, the FFA mechanism of action is not yet understood. The involvement of somatostatin (SRIH) is controversial, and in vitro experiments suggest a direct effect of FFA on the pituitary. In sheep it is possible to collect hypophysial portal blood and quantify SRIH secretion in hypophysial portal blood under physiological conscious and unstressed conditions. In this study we determined the effects of FFA (Intralipid and heparin) infusion on peripheral GH and portal SRIH levels in intact rams chronically implanted with perihypophysial cannula and in rams actively immunized against SRIH to further determine SRIH-mediated FFA effects on GH axis. Immediately after initiation of Intralipid infusion, we observed a marked increase in the FFA concentration (2160 +/- 200 vs. 295 +/- 28 nmol/ml; P < 0.01) as well as a significant decrease in basal GH secretion (1.8 +/- 0.1 vs. 2.5 +/- 0.3 ng/ml; P < 0.05) and a drastic reduction of the GH response to i.v. GH-releasing hormone injection (4.8 +/- 0.7 ng/ml in FFA group vs. 35.8 +/- 9.7 ng/ml in saline group; P < 0.01). No change in plasma insulin-like growth factor I levels was observed. During the first 2 h of infusion, the GH decrease observed was concomitant with a significant increase in portal SRIH levels (22.1 +/- .2 vs. 13 +/- 1.6 pg/ml; P < 0.01). In rams actively immunized against SRIH, the effect of FFA on basal GH secretion was biphasic. During the first 90 min of infusion, the decrease in GH induced by FFA was significantly blunted in rams actively immunized against SRIH (57 +/- 9% for immunized rams vs. 23.5 +/- 2.5% for control rams). This corresponds to the period of increased SRIH portal levels. After this first 90-min period, no difference was seen between control and immunized rams. Our results show that FFA exert their inhibitory action on the GH axis at both pituitary and hypothalamic levels, the latter mainly during the first 90 min, through increased SRIH secretion.

Endotoxin injection increases growth hormone and somatostatin secretion in sheep.

Endotoxin has been shown to stimulate GH secretion in human and sheep. However, changes in hypothalamic neurohormones involved in the GH regulation by endotoxin have never been studied in vivo. In sheep it is possible to collect hypophysial portal blood (HPB) and quantify GH-releasing hormone (GHRH) and somatostatin (SRIH) secretion under physiological conditions. The purpose of this study was to determine the effect of an acute i.v. endotoxin administration on the secretion of these peptides in sheep. Endotoxin induced a sustained increase of GH (x6.2 +/- 1.3) in intact rams. This stimulation was delayed and less marked when compared with the hypothalamic-pituitary-adrenal axis. Surprisingly, the GH increase was associated with an important rise of jugular (x10.6 +/- 2.4) and portal (x7.9 +/- 3) SRIH levels, without a significant GHRH increase. To determine if the portal SRIH increase was a consequence of an increased short feedback of GH, we studied GH response to endotoxin after a previous GHRH injection to deplete the pituitary pools of GH. In that case, despite the absence of increase of GH after endotoxin treatment, SRIH levels was markedly increased. For the first time we have observed an experimental situation in sheep with a simultaneous and closed amplitude increase in jugular and portal SRIH. The source of jugular SRIH is likely the gastrointestinal tract and the increased jugular SRIH release in systemic circulation might be in part responsible for the increase of hypophysial portal SRIH. Ultimately our results show that endotoxin induced a complex reaction at multiple levels with a specific increase in both portal and peripheral SRIH levels. The surprising association of a lack of change in GHRH release and an increased secretion of SRIH with the increase of GH suggests that the effect of endotoxin on GH axis is mainly a pituitary one. The selective blockade of somatostatin should be useful for a better knowledge of the role of SRIH stimulation in the physiopathology of septic shock.

Species differences between male rat and ram pituitary somatostatin receptors involved in the inhibition of growth hormone secretion.

The sheep is a valuable model in which to study GH neuroregulation as its pattern of GH secretion is very close to that in humans. Furthermore, important differences in somatostatin (SRIH) action between rats and sheep have been found previously. Our goal was to compare in male rat and ram pituitaries the binding characteristics of somatostatin receptors and the effect of SRIH and 17 analogues on GH release. Using radioautography, SRIH binding was seen to be evenly distributed over the anterior pituitary of both species. In the binding assay, binding sites were three times more concentrated in rats than in sheep. Important interspecies differences in the action of SRIH and its analogues were found: they inhibited GH at lower concentrations in rats than in sheep. Seven peptides displayed greater inhibitory ability in sheep than in rats while three were more potent in rats. Agonistic potencies to inhibit GH release in rats were correlated with somatostatin receptors subtype 2 (sst2) affinities. Our data confirm and extend the quantitative differences between rat and sheep in SRIH inhibitory action on GH secretion and confirm that ligand-binding properties of a given receptor subtype cannot be extrapolated across species.