Somatostatin Agonist Pasireotide Inhibits Exercise-Stimulated Growth in the Male Siberian Hamster (Phodopus sungorus).

The Siberian hamster (Phodopus sungorus) is a seasonal mammal, exhibiting a suite of physiologically and behaviourally distinct traits dependent on the time of year and governed by changes in perceived day length (photoperiod). These attributes include significant weight loss, reduced food intake, gonadal atrophy and pelage change with short-day photoperiod as in winter. The central mechanisms driving seasonal phenotype change during winter are mediated by a reduced availability of hypothalamic triiodothyronine (T3), although the downstream mechanisms responsible for physiological and behavioural changes are yet to be fully clarified. With access to a running wheel (RW) in short photoperiod, Siberian hamsters that have undergone photoperiod-mediated weight loss over-ride photoperiod-drive for reduced body weight and regain weight similar to a hamster held in long days. These changes occur despite retaining the majority of hypothalamic gene expression profiles appropriate for short-day hamsters. Utilising the somatostatin agonist pasireotide, we recently provided evidence for an involvement of the growth hormone (GH) axis in the seasonal regulation of bodyweight. In the present study, we employed pasireotide to test for the possible involvement of the GH axis in RW-induced body weight regulation. Pasireotide successfully inhibited exercise-stimulated growth in short-day hamsters and this was accompanied by altered hypothalamic gene expression of key GH axis components. Our data provide support for an involvement of the GH axis in the RW response in Siberian hamsters.

[Ectopic acromegaly due to a bronchial carcinoid]. / Acromegalia ectópica por carcinoide bronquial.

Tumours that cause ectopic acromegaly can do so through the secretion of GH or GHRH. One hundred cases of ectopic acromegaly due to secretion of GHRH have been described. Given the rarity of this pathology, we present a clinical case with the aim of contributing our diagnostic-therapeutic experience and the subsequent follow-up. We present the case of a patient with acromegaloid physical features that had evolved over several years. Concomitantly, he also presented other accompanying symptoms that were suggestive of a possible bronchial origin. Facing the clinical suspicion of acromegaly, we opted to confirm it biochemically and subsequently through image study. A hypophysary origin was ruled out, so we carried out screening for a bronchial neuroendocrine and/or gastrointestinal tumor as they are the most frequent localizations. The treatment of choice was surgical resection.

PLGA microsphere-mediated growth hormone release hormone expression induces intergenerational growth.

To improve animal growth, growth hormone-releasing hormone (GHRH) expression vectors that maintain constant GHRH expression can be directly injected into muscles. To deliver the GHRH expression vectors, biodegradable microspheres have been used as a sustained release system. Although administering GHRH through microspheres is a common practice, the intergenerational effects of this delivery system are unknown. To investigate the intergenerational effects of polylactic-co-glycolic acid (PLGA) encapsulated plasmid-mediated GHRH supplements, pCMV-Rep-GHRH microspheres were injected into pregnant mice. Growth and expression of GHRH were measured in the offspring. RT-PCR and immunohistochemistry reveal GHRH expression 3-21 days post-injection. The proportion of GH-positive cells in the GHRH treated offspring was 48.2% higher than in the control group (P < 0.01). The GHRH treated offspring were 6.15% (P < 0.05) larger than the control offspring. At day 49 post-injection, IGF-I serum levels were significantly higher in the treatment group than in the control group. This study confirms that intramuscular expression of GHRH mediated by PLGA microspheres significantly enhances intergenerational growth.

Inhibition of estrogen receptor positive and negative breast cancer cell lines with a growth hormone-releasing hormone antagonist.

GHRH antagonists have been shown to inhibit growth of various human cancer cell lines xenografted into nude mice including estrogen receptor negative human breast cancers. Previous observations also suggest that GHRH locally produced in diverse neoplasms including breast cancer might directly affect proliferation of tumor cells. In the present study we demonstrate that a novel highly potent GHRH antagonist JMR-132 strongly inhibits the proliferation of both estrogen receptor negative SKBR 3 and estrogen receptor positive ZR 75 human breast cancer cell lines in vitro. The proliferation in vitro of ZR 75 and SKBR 3 was increased after direct stimulation with GHRH(1-29)NH2. The GHRH antagonist JMR-132 had a significant antiproliferative activity in the absence of GHRH and nullified the proliferative effect of GHRH in these cell lines. SKBR 3 and ZR 75 expressed the GHRH ligand as well as the pituitary type of GHRH-receptor, which likely appears to mediate the antiproliferative mechanisms in these cell lines. These in vitro results suggest that JMR-132 is a potent inhibitor of breast cancer growth, independent of the estrogen receptor status. Further investigations on the combination treatment with endocrine agents affecting the estrogen pathway and GRHR antagonists are needed in order to improve the treatment of breast cancer.

Acromegaly caused by a growth hormone-releasing hormone secreting carcinoid tumour of the lung: the effect of octreotide treatment.

In acromegaly, the overproduction of growth hormone is usually caused by a pituitary adenoma. We report a 74-year-old woman with acromegaly caused by ectopic overproduction of growth hormone-releasing hormone (GHRH), a rare diagnosis. The GHRH appeared to be produced by a carcinoid tumour of the lung. Treatment with monthly long-acting octreotide resulted in a reduction in the symptoms and normalisation of the insulin-like growth factor-I, which has been maintained for more than two years now. A review of literature concerning causes and treatment of ectopic GHRH-producing tumours is presented.

Gene expression in arcuate nucleus-median eminence of rats treated with leptin or ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) and leptin are cytokine-like% hormones and act on their corresponding receptors in the hypothalamic arcuate nucleus (ARC). The present study was designed to assess effects of intracerebroventricular (ICV) injection of leptin and CNTF on gene expression in micropunched hypothalamic arcuate nucleus-median eminence (ARC-ME) complex samples from rats. Male Sprague Dawley rats were implanted with lateral cerebroventricular cannulas for administration of control, 10 microg/d leptin or 5 microg/d CNTF for four days. Real-time Taqmantrade mark RT-PCR was used to quantitatively compare the mRNA levels of selected genes in the ARC-ME complex. Leptin and CNTF increased ARC-ME mRNA levels of signal transducer and activator of transcription 3 (STAT3) by 64.5 and 124.7% (p<0.01), suppressor of cytokine signaling 3 (SOCS3) by 258.9 and 1063.9% (p<0.01), cocaine and amphetamine regulated transcript (CART) by 102.7 and 123.1% (p<0.01), and proopiomelanocortin (POMC2) by 374.1 and 264.9% (p<0.01), respectively. Leptin increased growth hormone releasing hormone (GHRH) by 309.9% (p<0.01), while CNTF increased janus kinase 2 (JAK2) mRNA by 31.7% (p<0.01) and decreased gonadotropin releasing hormone 1 (GNRH1) by 59.7% (p<0.01), mitogen activated protein kinase 1 (MAPK1) by 19.4% (p<0.05) and tyrosine hydroxylase (TH) by 74.5% (p<0.05). Significant reduction in daily food intake and body weights by both the treatments was observed. Also, decrease in weights of fat pads was concomitant with lowered serum insulin and leptin levels. Our findings show that leptin and CNTF engage both convergent and divergent pathways involved in feeding, cellular signaling, inflammation, and other related regulatory systems.

Expression of growth hormone-releasing hormone (GHRH) and splice variant of GHRH receptors in normal mouse tissues.

Growth hormone-releasing hormone (GHRH) stimulates the production and release of growth hormone in the pituitary and induces cell proliferation in a variety of peripheral tissues and tumors. These extrapituitary effects of GHRH are in many cases mediated by a splice variant of GHRH receptor designated SV1 that differs from the pituitary GHRH receptor in a small portion of its amino-terminal region. While SV1 has been detected in several primary tumors and many cancer cell lines its expression in normal tissues remains unclear. In this study we report the results of an immunohistochemical analysis for SV1 and GHRH expression in normal mouse tissues. For the detection of SV1 immunoreactivity we used a polyclonal antiserum against segments 1-25 of the SV1 receptor protein. Mouse heart, colon, lungs, small intestine, stomach and kidneys exhibited increased SV1 immunoreactivity. These tissues were also positive for GHRH expression, however, tissues such as the endometrium were positive only for GHRH and not for SV1 expression. On the contrary, testis were positive for SV1 and not for GHRH expression. These results indicate that SV1 may play a role in normal physiology.

Genetics and proteomics of pituitary tumors.

Genetics and proteomics determine structure and function of normal tissues, and the molecular alterations that underlie tumorigenesis result in changes in these aspects of tissue biology in neoplasms. We review the known genetic alterations in pituitary tumors. These include the oncogenic Gsalpha protein (GSP)-activating mutations, and pituitary tumor-derived fibroblast growth factor receptor-4 (ptd-FGFR4), as well as tumor suppressor gene mutations associated with multiple endocrine neoplasia type 1 (MEN1). Other candidates identified from expression profiling include pituitary tumor-transforming gene (PTTG), GADD45, and bone morphogenic protein (BMP)4. Proteomic changes in pituitary tumors include classical alterations identified by immunohistochemistry as well as epigenetic reductions in p27. The underlying mechanisms for dysregulated cell adhesive molecules including cadherins and FGFRs are reviewed. The combined use of genetic and proteomic approaches will enhance novel drug therapeutic development.

Identification of ghrelin and its receptor in neurons of the rat arcuate nucleus.

Ghrelin, an acylated peptide originally identified in rat stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), stimulates both food intake and growth hormone (GH) secretion. Ghrelin is predominantly synthesized by a subset of endocrine cells in the oxyntic gland of human and rat stomach. Previous studies using immunohistochemistry have shown that ghrelin is also present in the hypothalamic arcuate nucleus, a region critical for the control of feeding and GH secretion, but its expression pattern in this region and the details of its molecular form has yet to be clarified. In this report, we examined the presence of ghrelin in the arcuate nucleus using reverse-phase liquid chromatography combined with radioimmunoassay (RIA) and immunohistochemistry. Neurons in the arcuate nucleus were observed to react positively to ghrelin antibodies. In addition, we confirmed the existence of ghrelin mRNA expression using the reverse-transcription polymerase chain reaction (RT-PCR). We also observed the colocalization of GHS-R with neuropeptide Y (NPY) and growth-hormone-releasing hormone (GHRH) in the arcuate nucleus. The present study clearly indicates that ghrelin is synthesized in the arcuate nucleus, which will further our understanding of ghrelin's actions in the central nervous system, including feeding behavior and GH secretion.

Stepwise posttranslational processing of progrowth hormone-releasing hormone (proGHRH) polypeptide by furin and PC1.

Through a posttranslational processing mechanism, pro-growth hormone releasing hormone (proGHRH) gives rise to an amidated GHRH molecule, which in turn stimulates the synthesis and release of growth hormone. We have previously proposed a model for the biochemical processing of proGHRH [Nillni et al. (1999), Endocrinology 140, 5817-5827]. We demonstrated that the proGHRH peptide (10.5 kDa, 104 aa) is first processed to an 8.8 kDa intermediate form that is later cleaved to yield two products: the 5.2 kDa GHRH and the 3.6 kDa GHRH-RP. However, the proteolytic enzymes involved in this process are unknown. Therefore, in this study we determined which proconverting enzymes are involved in this process. We transfected different constructs in cell lines carrying different PC enzymes followed by analysis of the peptide products after metabolic labeling or Western blots. We found that in the absence of furin (LoVo cells) or CHO cells treated with BFA, only one moiety was observed, and that corresponds to the same electrophorectic mobility to the GHRH precursor. This finding strongly supports an initial role for furin in the processing of proGHRH. The results from transfections with preproGHRH alone or double or triple transfections with PC1 and PC2 in AtT-20, GH3, and GH4C1 cells indicated that PC1 is the primary enzyme involved in the generation of GHRH peptide from the 8.8 kDa intermediate form. We found that AtT-20 cells (high PC1, very low PC2) were able to generate GHRH. However, GH3 cells (high PC2, but not PC1) were able to process the 8.8 kDa peptide to GHRH only after the cotransfection with the PC1 enzyme. Transfections with preproGHRH-GFP and preproGHRH-V5 provided similar results in all the cell lines analyzed. These data support the hypothesis that proGHRH is initially cleave by furin at preproGHRH29-30, followed by a second cleavage at preproGHRH74 primarily by PC1 to generate GHRH and GHRH-RP peptides, respectively.

Growth hormone-releasing hormone and extra-pituitary tumorigenesis: therapeutic and diagnostic applications of growth hormone-releasing hormone antagonists.

Growth hormone-releasing hormone (GHRH) regulates growth hormone release from the pituitary. However, in addition to this neuroendocrine action, much evidence implies an additional role for GHRH in carcinogenesis in non-pituitary tissues. This role of GHRH in cancer development appears to be due to the operation of several mechanisms, which involve the regulation of the growth hormone-dependent hepatic insulin-like growth factor I (IGFI) production, tumoural IGF-I and IGF-II secretion and direct action of GHRH on tumour cells by autocrine and/or paracrine pathways. This review summarises the available information regarding the role of GHRH in tumorigenesis with special emphasis on the direct action of GHRH in primary and experimental cancers.

Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers.

The expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors in LNCaP, MDA-PCa-2b and PC-3 human prostate cancers grown in nude mice was investigated by RT-PCR. The expression of mRNA for GHRH was detected in LNCaP and PC-3, but not in MDA-PCa-2b prostatic carcinoma. RT-PCR analyses of mRNA isolated from LNCaP, MDA-PCa-2b and PC-3 cancers, revealed the presence of 720 and 566 bp products, corresponding to SV(1) and SV(2) isoforms of GHRH receptors. In PC-3 tumor membranes a radiolabeled GHRH antagonist [125I]-JV-1-42 was bound to one class of high-affinity binding sites (K(d)=1.81+/-0.47 nM) and maximum binding capacity of 332.7+/-27.8 fmol/mg membrane protein. The in vivo uptake of [125I]-JV-1-42 was observed in all xenografts of human prostate cancer, the tracer accumulation being the highest in PC-3 tumors. These results indicate that GHRH and SVs of its receptors, different from those found in the pituitary, are present in experimental human prostate cancers and may form a local mitogenic loop. The antiproliferative effects of GHRH antagonists on growth of prostate cancer could be exerted in part by an interference with this local GHRH system.

Direct modification of somatotrope function by long-term leptin treatment of primary cultured ovine pituitary cells.

Leptin is produced primarily in adipocytes and regulates body energy balance. A close link between leptin and pituitary hormones, including GH, has been reported. The mechanisms employed by leptin to influence somatotropes are not clear, however. Here we report a direct action of recombinant ovine leptin on primary cultured ovine somatotropes by analyzing the levels of mRNA encoding for GH or the receptors for GHRH (GHRH-R) and GH-releasing peptides (GHRP). Treatment of ovine somatotropes with leptin (10(-7)-10(-9) M) for 1-3 d reduced the mRNA levels encoding GH and GHRH-R, but increased GHRP receptor mRNA levels in a time- and dose-dependent manner. Three-day treatment of cells with leptin decreased the GH response to GHRH stimulation, but the GH response to GHRP-2 stimulation was increased. The combined effect of GHRH and GHRP-2 on GH secretion was not altered by treatment of the cells with leptin. These results demonstrated a direct action of leptin on ovine pituitary cells, leading to a reduced sensitivity of somatotropes to GHRH. It is also suggested that GHRP may be useful to correct the decrease in GHRH-induced GH secretion by leptin.

A potential autocrine pathway for growth hormone releasing hormone (GHRH) and its receptor in human prostate cancer cell lines.

BACKGROUND: Recent studies have shown that GHRH antagonists inhibit prostate tumour growth and IGF-II production both in vivo and in vitro. The mechanism underlying these observations is unknown, but may involve an interaction with a prostatic GHRH receptor (GHRH-R), raising the possibility of an autocrine pathway for the GHRH axis in the prostate. METHODS: GHRH and GHRH-R mRNA expression was examined by RT-PCR in human prostate cancer cell lines, and the authenticity of PCR products was confirmed by Southern analysis and cDNA sequencing. Immunohistochemical techniques were used to examine the expression of GHRH protein in prostate cancer cell lines. RESULTS: GHRH-R (mRNA) and GHRH (mRNA and protein) are co-expressed in the ALVA-41, DU145, LNCaP and PC3 human prostate cancer cell lines. CONCLUSIONS: These observations suggest the presence of an intact prostatic GHRH autocrine pathway which may stimulate prostate cell proliferation. This pathway may be disrupted by GHRH antagonists.

Hypertrophic pulmonary osteoarthropathy associated with non-small cell lung cancer demonstrated growth hormone-releasing hormone by immunohistochemical analysis.

Hypertrophic pulmonary osteoarthropathy (HPO) associated with non-small cell lung cancer in a 58-year-old man was accompanied by an elevated serum level of growth hormone (GH). HPO rapidly disappeared after resection of the primary tumor and the elevation of serum GH was resolved. Immunohistochemically the tumor contained growth hormone-releasing hormone (GHRH) but not GH. These findings suggest that the high serum GH level due to ectopic GHRH production in the tumor, was a contributing factor in HPO. This is the second reported case of non-small cell lung cancer which was immunohistochemically positive for GHRH associated with HPO.

Human uterine and ovarian expression of growth hormone-releasing hormone messenger RNA in benign and malignant gynecologic conditions.

OBJECTIVE: To determine uterine and ovarian expression of growth hormone-releasing hormone (GHRH) messenger RNA (mRNA) in benign and pathologic gynecologic states. DESIGN: Case-control study. SETTING: Tertiary-care academic department. PATIENT(S): Women undergoing hysterectomy for benign or malignant gynecologic conditions. INTERVENTION(S): Ovarian and uterine tissue was obtained for measurement of GHRH mRNA levels by reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURE(S): Levels of GHRH mRNA in normal tissues were compared with those in tissues with pathologic abnormalities. RESULT(S): Growth hormone-releasing hormone mRNA was detectable in the ovary, endometrium, myometrium, fallopian tubes, and placenta. Levels of GHRH mRNA were significantly increased in secretory endometrium compared with proliferative endometrium. Hormone replacement therapy did not affect endometrial GHRH mRNA levels. Uterine myomas expressed similar levels of GHRH mRNA as normal myometrium. No changes in endometrial GHRH mRNA were detected in endometrial cancers compared with normal endometrium or myometrium obtained from the same patient; however, these levels were higher than those in noncancerous myometrial tissue obtained from other patients with benign gynecologic disease. In ovarian tissue, no differences in GHRH mRNA were found between premenopausal and postmenopausal women. Ovarian GHRH mRNA was significantly decreased in endometriotic cysts, whereas significantly greater GHRH expression occurred in ovarian cancer compared with normal ovarian tissue. CONCLUSION(S): Endometrial and ovarian GHRH gene transcription are altered in selective physiologic and pathologic states and are influenced by such factors as ovarian hormones. Because it is a growth factor, GHRH may promote endometrial proliferation and may be involved in the pathogenesis of ovarian and endometrial cancer and endometriosis.

Molecular cloning of growth hormone-releasing hormone/pituitary adenylyl cyclase-activating polypeptide in the frog Xenopus laevis: brain distribution and regulation after castration.

Pituitary adenylyl cyclase-activating peptide (PACAP) appears to regulate several neuroendocrine functions in the frog, but its messenger RNA (mRNA) structure and brain distribution are unknown. To understand the potential role of PACAP in the male frog hypothalamic-pituitary-gonadal axis, we cloned the frog Xenopus laevis PACAP mRNA and determined its distribution in the brain. We then analyzed the castration-induced alterations of mRNA expression for PACAP and its selective type I receptor (PAC1) in the hypothalamic anterior preoptic area, a region known to regulate reproductive function. The PACAP mRNA encodes a peptide precursor predicted to give rise to both GH-releasing hormone and PACAP. The deduced peptide sequence of PACAP-38 was nearly identical to that of human PACAP with one amino acid substitution. Abundant PACAP mRNA was detected in the brain, but not several other tissues, including the testis. In situ hybridization revealed strong expression of the PACAP gene in the dorsal pallium, ventral hypothalamus, and nuclei of cerebellum. PACAP mRNA signals were weak to moderate in the hypothalamic anterior preoptic area and were absent in the pituitary. Castration induced an increase in the expression of PACAP and PAC1 receptor mRNAs in the hypothalamic anterior preoptic area after 3 days. Replacement with testosterone prevented the castration-induced changes. These results provide a molecular basis for studying the physiological functions of PACAP in frog brain and suggest that PACAP may be involved in the feedback regulation of hypothalamic-pituitary-gonadal axis.