SSTR2 Antagonist Research Articles

SSTR2 Mediates the Inhibitory Effect of SST/CST on Lipolysis in Chicken Adipose Tissue.

Somatostatin shows an anti-lipolytic effect in both chickens and ducks. However, its molecular mediator remains to be identified. Here, we report that somatostatin type 2 receptor (SSTR2) is expressed at a high level in chicken adipose tissue. In cultured chicken adipose tissue, the inhibition of glucagon-stimulated lipolysis by somatostatin was blocked by an SSTR2 antagonist (CYN-154086), supporting an SSTR2-mediated anti-lipolytic effect. Furthermore, a significant pro-proliferative effect was detected in SST28-treated immortalized chicken preadipocytes (ICP-1), and this cell proliferative effect may be mediated through the MAPK/ERK signaling pathway activated by SSTR2. In summary, our results demonstrate that SSTR2 may regulate adipose tissue development by affecting the number and volume of adipocytes in chickens.

KS01.7.A ADAPTOR FITC CAR T-CELLS AND OCTO-FLUO ELICIT PROMISING ANTITUMOR ACTIVITY IN MENINGIOMA VIA SSTR2

Abstract BACKGROUND Effective treatment options for meningioma patients beyond surgical resection and radiotherapy are lacking. Somatostatin receptor (SSTR) 2 is expressed by most meningiomas. Here, we report pre-clinical and translational evidence for the activity of an SSTR2-targeted adaptor CAR T cell system in meningiomas. MATERIAL AND METHODS Tissue microarrays of meningioma (N=384) patient samples stained for SSTR2. Fluorescein-linked, octameric cyclic peptide SSTR2 antagonist OCTO-FLUO (kindly provided by Philogen, Otelfingen, Switzerland). Adaptor FITC CAR T-cells generated by lentiviral transduction of healthy donor T-cells. Flow cytometry-based killing assays in vitro in SSTR2-expressing meningioma (Ben-Men-1 IOMM-Lee) cells, and ex vivo utilizing freshly dissociated surgical meningioma specimens, by co-incubation with OCTO-FLUO and adaptor FITC CAR T-cells. Orthotopic growth and mouse survival following subdural implantation of IOMM-Lee cells, genetically engineered to express luciferase, i.v. treated with OCTO-FLUO and intratumoral injection of CAR T-cells. RESULTS SSTR2 expression was overall heterogeneous and was high or intermediate in most World Health Organization (WHO) grade 2 (87.8 %) or grade 3 (87.5 %) meningiomas. In vitro, 10 nM was determined as the optimal concentration of OCTO-FLUO, yielding tumor cell killing within 72 hours at effector-target cell (ET) ratios as low as 1:100 in Ben-Men-1 (CAR vs untransduced T-cells [UTT] 47.9 % vs 4.7 %, p < 0.001) and IOMM-Lee (CAR vs UTT 40.2 % vs 4.9 %, p < 0.001) cells. Tumor growth inhibition by the adaptor FITC CAR T-cell system in vivo was confined to SSTR2 expressing cells and was paralleled by prolonged survival compared to UTT in IOMM-Lee (hazard ratio [95% confidence interval], 0.3 [0.1 - 0.9], p=0.030). Preliminary ex vivo killing assays employing freshly dissociated patient samples yielded specific lysis-rates in the range of 15-20% (ET 1:1) at 12 hours. Updated ex vivo results employing optimized protocols will be presented at the conference. CONCLUSION Targeting SSTR2 positive meningioma cells with an adaptor FITC CAR T-cell system is feasible. Syngeneic murine models and the use of adaptor molecules directed against murine SSTR2 will be required to explore secondary immune reactions and off-tumor effects prior to proceeding to early phase clinical trials.

The usefulness of [68Ga]Ga-DOTA-JR11 PET/CT in patients with meningioma: comparison with MRI.

Clinical studies of PET imaging using SSTR2 agonists have demonstrated high accuracy and correlation with SSTR2 expression in meningiomas. However, the usefulness of the SSTR2 antagonist with [68Ga]Ga-DOTA-JR11 is uncertain. To evaluate the diagnostic performance of [68Ga]Ga-DOTA-JR11 PET/CT and to clarify tumor characteristics in patients with suspected meningiomas. Patients with suspected de novo or recurrent meningioma in complex locations or atypical images were enrolled from August 2021 to October 2022 in prospective study. All patients underwent contrast-enhanced MRI (CE-MRI), [68Ga]Ga-DOTA-JR11 PET/CT, and histopathological evaluation. Tumor uptake of [68Ga]Ga-DOTA-JR11 was measured by SUVmax and tumor-endocranium ratio (TBR). Diagnostic performance was compared between PET and MRI. Of 36 (50.0 ± 13.0years of age, 20 women) patients, 32 were histopathologically confirmed meningiomas and four with other tumors. [68Ga]Ga-DOTA-JR11 uptake was significantly higher in meningioma patients than in those with other tumors (SUVmax: 13.6 ± 7.7 vs. 5.2 ± 3.0, P < 0.001; TBR: 64.2 ± 27.7 vs. 25.0 ± 18.9, P = 0.001). [68Ga]Ga-DOTA-JR11 PET/CT detected 31 meningiomas, while CE-MRI detected 17 meningiomas of 25 initial diagnosis and 11 recurrent tumors; [68Ga]Ga-DOTA-JR11 PET had an incremental diagnostic value of 24% (6/25) over MRI in the group of initial diagnosis. There was no statistically significant difference in diagnostic efficacy between PET and MRI (P = 0.45) for all 36 patients. In skull base meningiomas, PET provided a more definitive diagnosis of pituitary involvement (in 12, not in12), compared to MRI (in eight, possible in six, possible not in six, not in four). PET revealed bone involvement in all 14 patients proven by pathology, while MRI identified only 11. [68Ga]Ga-DOTA-JR11 PET/CT provided high image quality and presented an ideal diagnostic performance in detecting meningioma and evaluating the involvement of the pituitary and bone. The study provides valuable evidence for the use of [68Ga]Ga-DOTA-JR11 PET/CT as a complementary imaging modality to CE-MRI in the evaluation of meningiomas.

Changes in dorsal root ganglion CGRP expression in mouse pinch nerve injury model: Modulation by Somatostatin type-2 receptor

IntroductionOur previous work has shown that somatostatin effectively inhibits neuropathic pain by activating its type 2 receptor (SSTR2) in the dorsal root ganglion (DRG) and spinal cord of mice. However, the underlying mechanism of this activation has not been elucidated. MethodsTo explore further mechanisms, we examined pain behavior and the expression of neuropeptides such as calcitonin gene-related peptide (CGRP) in dorsal root ganglion neurons(DRGs) as well as the changes of the number of CGRP-IR DRGs in the mouse model of sciatic pinch nerve injury. ResultsIn this model, the number of medium and small DRG neurons in ipsilateral CGRP-IR was slightly increased, but not significantly, compared with sham animals at 3, 7, and 9 days after pinch nerve injury. This correlated with the behavioral readouts of hypersensitivity at the same time points. However, the magnitude of the painful behavior (Autotomy) was observed after application of SSTR2 antagonist (CYN154806, 5 mg/kg) in the injured nerve groups compared to the saline-treated injured group as well as the sham-operated group. Following pinch nerve injury, there was a significant decrease in the number of ipsilateral CGRP-IR small and medium DRG neurons in SSTR2 antagonist (anti-SSTR2)- but not saline-treated mice. These data also correlated with painful behavioral readouts where hypersensitivity was significantly increased by anti-SSTR2 but not saline treatment. Discussion/conclusionIn all, application of the SSTR2 antagonist to the pinched sciatic nerve suppressed CGRP expression and aggravated painful behavior, suggesting that CGRP expression in DRG neurons can be an important component of the pain mechanism and an indicator of pain behavior.

Role of Basolateral Amygdalar Somatostatin 2 Receptors in a Rat Model of Chronic Anxiety

Repeated exposure to stress has been implicated in inducing chronic anxiety states. Stress related increases in anxiety responses are likely mediated by activation of corticotropin-releasing factor receptors (CRFR) in the amygdala, particularly the basolateral amygdala (BLA). Within the BLA, acute injections of the CRFR agonist urocortin 1 (Ucn1) leads to acute anxiety, whereas repeated daily injections of subthreshold-doses of Ucn1 produces a long-lasting, persistent anxiety-like phenotype, a phenomenon referred to as Ucn1-priming. Relative gene expressions from the BLA of vehicle and Ucn1-primed rats were analyzed with quantitative RT-PCR using a predesigned panel of 82 neuroscience-related genes. Compared to vehicle-primed rats, only expression of the somatostatin receptor 2 gene (Sstr2) was significantly reduced in the BLA of Ucn1-primed rats. The contribution of Sstr2 on an anxiety phenotype was tested by injecting a Sstr2 antagonist into the BLA in un-primed rats. The Sstr2 antagonist increased anxiety-like behavior. Notably, pretreatment with Sstr2 agonist injected into the BLA blocked anxiety-inducing effects of acute Ucn1 BLA-injections and delayed anxiety expression during Ucn1-priming. However, concomitant Sstr2 agonist pretreatment during Ucn-1 priming did not prevent either the development of a chronic anxiety state or a reduction of BLA Sstr2 expression induced by priming. The data demonstrate that the persistent anxiety-like phenotype observed with Ucn1-priming in the BLA is associated with a selective reduction of Sstr2 gene expression. Although Sstr2 activation in the BLA blocks acute anxiogenic effects of stress and down-regulation of BLA Sstr2, it does not suppress the long-term consequences of prolonged exposure to stress-related challenges.

Direct labeling of a somatostatin receptor antagonist via peptide cyclization with Re, 99mTc and 186Re metal centers: Radiochemistry and in vitro evaluation

IntroductionWith the long-term goal of developing a diagnostic (99mTc) and therapeutic (186Re) agent pair for targeting somatostatin receptor (SSTR)-positive neuroendocrine tumors (NETs), we developed novel metal-cyclized peptides through direct labeling of the potent SSTR2 antagonist Ac-4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH2 (1) with Re (in Re-1), 99mTc (in [99mTc]Tc-1) and 186Re (in [186Re]Re-1). MethodsRe-1 was characterized by LC-ESI-MS and HR-ESI-MS and was tested for receptor affinity in SSTR-expressing cells (AR42J). Radiolabeling of the peptide was achieved via ligand exchange from 99mTc-labeled glucoheptonate or [186Re]ReOCl3(PPh3)2, yielding [99mTc]Tc-1 or [186Re]Re-1, respectively. In vitro stability of [99mTc]Tc-1/[186Re]Re-1 in PBS (10 mM) at pH 7.4 and 37 °C was determined by HPLC analysis. Moreover, [99mTc]Tc-1 stability was tested in cysteine (1 mM) and rat serum under the same conditions. ResultsRe-1 consisted of two isomers, confirmed by LC-ESI-MS, with good SSTR2 affinity (IC50 = 43 ± 6 nM). Optimization of the 99mTc labeling through varying reaction parameters such as pH, reaction time, and Sn2+ and ligand concentrations resulted in high radiochemical yield (RCY ≥92%). Similarly, [186Re]Re-1 was prepared in reasonable RCY (≥50%). Both 99mTc/186Re-tracers consisted of two product isomers as identified by HPLC co-injection with Re-1. While [99mTc]Tc-1 was sufficiently stable in vitro (≥71% intact through 4 h in PBS, cysteine and rat serum), [186Re]Re-1 exhibited more moderate in vitro stability (58% intact after 1 h in PBS). ConclusionsNovel 99mTc/186Re-cyclized SSTR2 antagonist peptides were synthesized and characterized using the Re-cyclized analogue as a reference. Due to the nanomolar SSTR2 affinity of Re-1 and good in vitro stability of [99mTc]Tc-1, the latter shows early promise for development as a radiodiagnostic agent for SSTR-expressing NETs. Advances in knowledge and implications for patient careThe 99mTc-cyclized complex showed promising in vitro properties, and future in vivo studies will determine the potential for translating such a design into the human clinic.

Inhibition of ASIC-Mediated Currents by Activation of Somatostatin 2 Receptors in Rat Dorsal Root Ganglion Neurons.

Somatostatin (SST) and its analogues like octreotide (OCT) have analgesic effect on a variety of pain through peripheral SST receptors (SSTRs). However, the precise molecular mechanisms have not yet been fully elucidated. This research aimed to identify possible antinociceptive mechanisms, showing functional links of the SSTR2 and acid-sensing ion channels (ASICs). Herein, we reported that OCT inhibited the electrophysiological activity of ASICs in rat dorsal root ganglia (DRG) neurons. OCT concentration-dependently decreased the peak amplitude of acid-evoked inward currents, which were mediated by ASICs. OCT shifted concentration-response curve to protons downwards, with a decrease of 36.53 ± 5.28% in the maximal current response to pH4.5 in the presence of OCT. OCT inhibited ASIC-mediated currents through SSTR2, since the inhibition was blocked by Cyn 154806, a specific SSTR2 antagonist. The OCT inhibition of ASIC-mediated currents was mimicked by H-89, a membrane-permeable inhibitor of PKA, and reversed by internal treatment of an adenylyl cyclase activator forskolin or 8-Br-cAMP. OCT also decreased the number of action potentials induced by acid stimuli through SSTR2. Finally, peripheral administration of 20μM OCT, but not 2μM OCT, significantly relieved nociceptive responses to intraplantar injection of acetic acid in rats. This occurred through local activation of SSTR2 in the injected hindpaw and was reversed following co-application of Cyn 154806. Our results indicate that activation SSTR2 by OCT can inhibit the activity of ASICs via an intracellular cAMP and PKA signaling pathway in rat DRG neurons. These observations demonstrate a cross-talk between ASICs and SSTR2 in peripheral sensory neurons, which was a novel peripheral analgesic mechanism of SST and its analogues.

The role of somatostatin and its receptors (sstr2, sstr5) in the contractility of gilt inflamed uterus

We studied the inflammation effect on somatostatin receptors subtypes 2 (sstr2) and 5 (sstr5) expression in myometrium and somatostatin influence alone or with sstr2 and sstr5 antagonists on the contractility of gilt inflamed uterus. On day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) were injected into uterine horns. In the control pigs (CON group), only laparotomy was performed. Eight days later, in the E.coli group developed severe acute endometritis. In this group, myometrial sstr2 mRNA expression lowered and protein expression increased compared to other groups. Compared to period before somatostatin administration, somatostatin did not change tension in myometrium and endometrium/myometrium of three groups, reduced amplitude and frequency in the CON and SAL groups, and increased amplitude and decreased frequency in the E.coli group. In this group, amplitude was increased by somatostatin compared to other groups. In the CON and SAL groups, sstr2 eliminated inhibitory somatostatin effect on amplitude, while sstr5 antagonist reversed inhibitory somatostatin effect on amplitude. In the E.coli group, sstr2 antagonist reversed stimulatory somatostatin effect on amplitude, while in sstr5 antagonist presence stimulatory somatostatin effect was more deepened compared to somatostatin action alone. After using sstr2 antagonist more deepened inhibitory somatostatin effect on frequency in the CON and E.coli groups was found. Sstr5 antagonist partly eliminated inhibitory somatostatin effect on frequency in the SAL group. Summarizing, the uterine inflammation increases the myometrial sstr2 protein expression; somatostatin raises amplitude of the inflamed uterus acting by sstr2, while drops this parameter by sstr5.

Somatostatin Type 2 Receptor Antibody Enhances Mechanical Hyperalgesia in the Dorsal Root Ganglion Neurons after Sciatic Nerve-pinch Injury: Evidence of Behavioral Studies and Bax Protein Expression.

Our previous study has indicated that somatostatin potently inhibits neuropathic pain through the activation of its type 2 receptor (SSTR2) in mouse dorsal root ganglion and spinal cord. However, the underlying mechanism of this activation has not been elucidated clearly. The aim of this study is to perform the pharmacological studies on the basis of sciatic nerve-pinch mice model and explore the underlying mechanism involving SSTR2. On the basis of a sciatic nerve-pinch injury model, we aimed at comparing the painful behavior and dorsal root ganglion neurons neurochemical changes after the SSTR2 antibody (anti- SSTR2;5μl,1μg/ml) administration in the mouse. After pinch nerve injury, we found that the mechanical hyperalgesia and severely painful behavior (autotomy) were detected after the application of SSTR2 antibody (anti-SSTR2; 5μl, 1μg/ml) on the pinch-injured nerve. The up-regulated phosphorylated ERK (p-ERK) expression and the apoptotic marker (i.e., Bax) were significantly decreased in DRGs after anti-SSTR2 treatment. The current data suggested that inhibitory changes in proteins from the apoptotic pathway in anti-SSTR2-treated groups might be taking place to overcome the protein deficits caused by SSTR2 antibody and supported the new therapeutic intervention with SSTR2 antagonist for neuronal degeneration following nerve injury.

Design and development of the theranostic pair 177Lu‐OPS201/68Ga‐OPS202 for targeting somatostatin receptor expressing tumors

Radiolabeled somatostatin receptor (sstr) antagonists have shown superiority in different preclinical and clinical settings compared with the well-established and clinically used agonists for targeting sstr-expressing tumors, with regard to pharmacokinetics, tumor uptake, and retention. The theranostic pair 177 Lu-OPS201/68 Ga-OPS202, based on the sstr2 antagonist JR11 (Cpa-c[d-Cys-Aph(Hor)-d-Aph(Cbm)-Lys-Thr-Cys]-d-Tyr-NH2 ), is the most advanced pair of the antagonist family in terms of preclinical development and is currently under clinical evaluation. OPS201 and OPS202 share the same amino acid sequence (JR11) but feature different conjugated chelators needed for radiolabeling, DOTA for OPS201 and NODAGA for OPS202. In this review, the design and development of the peptidic analog, JR11, and the selection of chelators and radiometals that led to 177 Lu-OPS201/68 Ga-OPS202 are discussed. Furthermore, the preclinical evaluation of both radiolabeled analogs from bench to bedside and the clinical trials involving the theranostic pair are presented.

Somatostatin receptor targeting with hydrophilic [99mTc/186Re]Tc/Re-tricarbonyl NODAGA and NOTA complexes

IntroductionThe aim of this work was to develop diagnostic (99mTc) and therapeutic (186Re) agents for targeting somatostatin receptor (SSTR)-positive neuroendocrine tumors (NETs). In this regard, we evaluated in vitro complexes of the general formula [M(CO)3(L-sst2-ANT)] (M = 99mTc, 186Re), where L denotes NODAGA or NOTA and sst2-ANT denotes the potent SSTR2 antagonist 4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH2. Moreover, we assessed the in vivo properties of the 99mTc-complexes in an animal SSTR-tumor model. MethodsThe [99mTc]/[186Re][Tc/Re(OH2)3(CO)3]+ precursors were utilized to prepare the 99mTc/186Re-complexes, which were identified by HPLC co-injection with their natRe analogues. The tracers were challenged in vitro at 37 °C against cysteine and histidine in phosphate-buffered saline (pH 7.4) and in rat serum. Biodistribution and micro-SPECT/CT imaging studies of the 99mTc-tracers were performed in AR42J tumor-bearing female ICR SCID mice. ResultsThe 99mTc-complexes were prepared in high radiochemical yield (RCY > 90%, by HPLC), with lower RCY (≤30%) obtained for 186Re-complexes. Tracers remained intact in vitro and displayed low non-specific binding (10–25%) to rat serum proteins. Biodistribution of [99mTc]Tc-NODAGA-sst2-ANT revealed low tumor uptake (2.78 ± 0.27 %ID/g) at 1 h, while high tumor uptake (16.70 ± 3.32 %ID/g) was found for [99mTc]Tc-NOTA-sst2-ANT. Moderate to low tumor retention was observed for both tracers after 4 and 24 h. Tumor uptake for [99mTc]Tc-NOTA-sst2-ANT was receptor-mediated, as demonstrated by parallel SSTR blocking studies. Rapid renal clearance was observed for both tracers, and SPECT/CT images clearly delineated the tumors, in agreement with the biodistribution data. ConclusionsThe [99mTc]Tc-NOTA-sst2-ANT complex demonstrated high tumor uptake and rapid clearance in a SSTR-tumor mouse model, showing potential for further development. Advances in knowledge and implications for patient carePreclinical data support the feasibility of the [99mTc]Tc/[186Re]Re-NOTA/NODAGA labeling strategy for use in the development of theranostic radiopharmaceuticals for translation into the human clinic for targeting of SSTR-expressing NETs.

Somatostatin-Somatostatin Receptor 2 Signaling Mediates LH Pulse Suppression in Lactating Rats.

Follicular development and ovulation are profoundly suppressed during lactation in mammals. This suppression is suggested to be mainly due to the suckling-induced inhibition of kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and consequent inhibition of pulsatile GnRH/LH release. We examined whether central somatostatin (SST) signaling mediates the suckling-induced suppression of pulsatile LH secretion. SST has been reported to be expressed in the posterior intralaminar thalamic nucleus (PIL), where the suckling stimulus is postulated to be relayed to the hypothalamus during lactation. SST inhibitory receptors (SSTRs) are abundantly expressed in the ARC, where kisspeptin/neurokinin B/dynorphin A (KNDy) neurons are located. Histological and quantitative studies revealed that the suckling stimulus increased the number of SST-expressing cells in the PIL, and Sstr2 expression in the ARC. Furthermore, a central injection of an SSTR2 antagonist caused a significant increase in pulsatile LH release in lactating rats. Double labeling of Sstr2 and the neurokinin B gene, as a marker for ARC KNDy neurons, showed Sstr2 expression was abundantly detected in the ARC, but few KNDy neurons coexpressed Sstr2 in lactating rats. Taken together, these findings suggest the suckling-induced activation of SST-SSTR2 signaling mediates, at least in part, the suppression of pulsatile LH secretion during lactation in rats, probably via the indirect effects of SST on KNDy neurons. These results provide a new aspect on the role of central SST-SSTR signaling in understanding the mechanism underlying lactational anestrus.

GLP-1 Secretion Is Increased upon Blockade of the Somatostatin Receptor Subtype 2 and 5 Resulting in GLP-1 Receptor-Mediated Lowering of Blood Glucose in Mice

Somatostatin (SS) is a hormone inhibiting the secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1) by binding to 5 SS receptors (SSTr). However, which SSTrs involved is still unclear. We hypothesized that antagonizing the SSTrs on enteroendocrine L-cells may increase secretion of GLP-1 and via GLP-1 receptor mediated mechanism lead to lowering of blood glucose. To investigate the level of the SSTrs we did expression analysis on L-cells isolated from throughout the murine intestine (n=3). We perfused the proximal small intestine from mice (n=6-7) to investigate the impact of SSTr2 and 5 on GLP-1 secretion using intra-arterial administration of specific antagonists. Lastly we performed oral glucose tolerance tests (OGTT) on mice (n=5-8) after giving 1mg/ml SSTr2 or 5 antagonist with and without the GLP-1 receptor antagonist exendin(9-39) to investigate the role of these receptors on the glucose profile and to elucidate if their effect was mediated via GLP-1. The expression levels of the SSTrs showed that SSTr2 and especially 5 had the highest expression on L-cells throughout the intestine. The SSTr2 antagonist resulted in a increase in GLP-1 secretion from the perfused intestine (100nM, 4.96pM±0.56; 1µM, 14.55pM±1.4; 10µM, 16.46pM±1.18pM; all p&amp;lt;0.0001). The same doses of SSTr5 antagonist gave a more potent GLP-1 secretion in this setup (100nM, 5.82pM±0.7 p=0.0004; 1µM, 19.79pM±1.2 p&amp;lt;0.0001; 10µM, 48pM±2.6 p&amp;lt;0.0001). Both SSTr2 and SSTr5 antagonists significantly reduced the blood glucose during an OGTT compared to the control (p=0.00and p=0.0072 respectively). This reduction in blood glucose was significantly increased when adding exendin(9-39) together with either the SSTr2 or 5 antagonist (p&amp;lt;0.0001 for SSTr2, p=0.0036 for SSTr5). Our results demonstrate that inhibiting the SSTr2 and SSTr5 increases GLP-1 secretion which in turn may cause the lowering in blood glucose during an OGTT. Disclosure S.L. Jepsen: None. N.J. Wewer Albrechtsen: Speaker's Bureau; Self; Merck Sharp &amp; Dohme Corp.. Other Relationship; Self; Mercodia, Alpco. J. Pedersen: None. M.S. Engelstoft: None. C.F. Deacon: Other Relationship; Self; Merck &amp; Co., Inc., Eli Lilly and Company. Employee; Spouse/Partner; Merck Sharp &amp; Dohme Corp.. Stock/Shareholder; Spouse/Partner; Merck &amp; Co., Inc. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Zealand Pharma A/S. Speaker's Bureau; Self; Merck Sharp &amp; Dohme Corp., AstraZeneca. Research Support; Self; Danish Diabetes Academy, Novo Nordisk Foundation. Other Relationship; Self; Antag Therapeutics. Other Relationship; Spouse/Partner; Antag Therapeutics.

Evidence That Endogenous Somatostatin Inhibits Episodic, but Not Surge, Secretion of LH in Female Sheep

Two modes of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion are necessary for female fertility: surge and episodic secretion. However, the neural systems that regulate these GnRH secretion patterns are still under investigation. The neuropeptide somatostatin (SST) inhibits episodic LH secretion in humans and sheep, and several lines of evidence suggest SST may regulate secretion during the LH surge. In this study, we examined whether SST alters the LH surge in ewes by administering a SST receptor (SSTR) 2 agonist (octreotide) or antagonist [CYN154806 (CYN)] into the third ventricle during an estrogen-induced LH surge and whether endogenous SST alters episodic LH secretion. Neither octreotide nor CYN altered the amplitude or timing of the LH surge. Administration of CYN to intact ewes during the breeding season or anestrus increased LH secretion and increased c-Fos in a subset GnRH and kisspeptin cells during anestrus. To determine if these stimulatory effects are steroid dependent or independent, we administered CYN to ovariectomized ewes. This SSTR2 antagonist increased LH pulse frequency in ovariectomized ewes during anestrus but not during the breeding season. This study provides evidence that endogenous SST contributes to the control of LH secretion. The results demonstrate that SST, acting through SSTR2, inhibits episodic LH secretion, likely acting in the mediobasal hypothalamus, but action at this receptor does not alter surge secretion. Additionally, these data provide evidence that SST contributes to the steroid-independent suppression of LH pulse frequency during anestrus.

Somatostatin stimulates intestinal NHE8 expression via p38 MAPK pathway

Diarrhea is a common manifestation of gastrointestinal disorders. Diarrhea-induced losses of fluid and electrolyte could lead to dehydration and electrolyte imbalances, resulting in significant morbidity and mortality, especially in children living in developing countries. Somatostatin, a peptide hormone secreted by D-cells, plays an important role in regulating motility and intestinal Na(+) absorption. Although octreotide, a somatostatin analog, is used to treat diarrhea, its mechanisms of action are unclear. Here we showed that octreotide increased brush-border membrane Na(+)/H(+) exchanger 8 (NHE8) expression in the small intestine to the exclusion of other NHEs that participate in Na(+) absorption. The same effect also occurred in human intestinal cells (Caco-2). We found that the increase of NHE8 expression by somatostatin required p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, the somatostatin receptor SSTR2 antagonist CYN154806 could abolish somatostatin-induced NHE8 expression and p38 MAPK phosphorylation. Thus our data provided the first concrete evidence indicating that somatostatin stimulates intestinal Na(+) absorption by increasing intestinal NHE8 expression through the SSTR2-p38 MAPK pathway.

Neuropeptides Galanin and Somatostatin Promote Neuronal Differentiation in Murine Subventricular Zone Cultures

Neuropeptides Galanin and Somatostatin Promote Neuronal Differentiation in Murine Subventricular Zone Cultures

Somatostatin inhibits insulin release via SSTR2 in hamster clonal β-cells and pancreatic islets

Somatostatin (SST) inhibits pancreatic endocrine secretion. It is generally accepted that SSTR2 and SSTR5 mediate the inhibition of glucagon and insulin release, respectively. The present study was performed to test the hypothesis that SSTR2, but not SSTR5, mediates SST-induced inhibition of insulin release in hamster β-cells. Both hamster clonal β-cells HIT-T15 and pancreatic islets were used to test this hypothesis. Both SST and a nonpeptide SSTR2 agonist L-779,976 (1–100 nM) inhibited insulin release from HIT-T15 and islets in a concentration-dependent manner. In contrast, nonpeptide agonists for SSTR1, 3, 4 and 5 at the highest concentration studied (1 μM) failed to inhibit insulin release. PRL-2903, a peptide SSTR2 antagonist (0.1–1 μM), antagonized SST-induced inhibition of insulin release in a concentration-dependent manner. Taken together, we conclude that, in hamster β-cells, SST inhibits insulin release via SSTR2 but not SSTR5.

Functional association of somatostatin receptor subtypes 2 and 5 in inhibiting human growth hormone secretion.

We previously demonstrated that somatostatin (SRIF)-induced inhibition of GH secretion from human pituitary cells is mediated through both the SRIF receptor (SSTR) 2 and 5 subtypes. The interplay between these two SSTR subtypes in regulating GH was therefore tested in primary human fetal pituitary cultures (18-30 wk gestation). GHRH (10 nM)-stimulated GH secretion (51% increase, P < 0.05) was suppressed equally by either SSTR2 or SSTR5-selective agonists (10 nM). GH suppression correlated with agonist affinity for their respective receptor subtypes. Combined addition of SSTR2- and SSTR5-specific agonists was synergistic for GH suppression, achieving 73% (P < 0.05) inhibition as compared with inhibition attained with SSTR2 (32%) and SSTR5 (34%) agonists used alone (P < 0.05). The SSTR2 selective antagonist BIM-23454 dose-dependently blocked SSTR2 but not SSTR5-induced suppression of GH secretion. BIM-23454 also completely reversed GH suppression in response to the combined activation of SSTR2 and SSTR5. The IC(50) for BIM-23454 reversal of agonist-induced GH suppression was 55 nM and 33 nM for two SSTR2 agonists, 45 nM and 40 nM for the combination of SSTR2 and SSTR5 agonists, respectively, and 45 nM for the SSTR2/SSTR5 agonist BIM-23244, all of which were similar to the affinity of BIM-23454 for SSTR2 (32 nM). These results suggest the following: 1) activation of both SSTR2 and SSTR5 induces a functional association of receptor subtypes, resulting in synergistic GH suppression; 2) BIM-23454 is a potent SSTR2-selective antagonist capable of reversing SRIF-induced GH suppression; and 3) the ability of a selective SSTR2 antagonist to inhibit the GH suppressing action of SSTR2 agonist alone, SSTR2/SSTR5 biselective agonists, or SSTR2 and SSTR5 agonists in combination support the concept of a functional interaction between somatotroph SSTR2 and SSTR5 subtypes in primary human fetal pituitary cells.

SSTR2 mediates the somatostatin-induced increase in intracellular Ca 2+ concentration and insulin secretion in the presence of arginine vasopressin in clonal β-cell HIT-T15

The effects of somatostatin (SRIF) are mediated through the seven transmembrane receptor family that signals via Gi/Go. To date, five distinct SRIF receptors have been characterized and designated SSTR1-5. We have characterized the SRIF receptor that mediates the increase in [Ca 2+] i and insulin secretion in HIT-T15 cells (Simian virus 40-transformed Syrian hamster islets) using high affinity, subtype selective agonists for SSTR1 (L-797,591), SSTR2 (L-779,976), SSTR3 (L-796,778), SSTR4 (L-803,087), SSTR5 (L-817,818) and PRL-2903, a specific SSTR2 antagonist. In the presence of arginine vasopressin (AVP), SRIF increased [Ca 2+] i and insulin secretion. Treatment with the SSTR2 agonist L-779,976 resulted in similar responses to SRIF. In addition, L-779,976 increased both [Ca 2+] i and insulin secretion in a dose-dependent manner. Treatment with L-779,976 alone did not alter [Ca 2+] i or basal insulin secretion. In the presence of AVP, all other SRIF receptor agonists failed to increase [Ca 2+] i and insulin secretion. The effects of SRIF and L-779,976 were abolished by the SSTR2 antagonist PRL-2903. Our results suggest that the mechanism underlying SRIF-induced insulin secretion in HIT-T15 cells be mediated through the SSTR2.

Somatostatin receptor subtypes 2 and 5 differentially affect proliferation in vitro of the human medullary thyroid carcinoma cell line tt.

Somatostatin and its receptors (SSTR1 to SSTR5) are expressed in normal human parafollicular C cells and medullary thyroid carcinoma (MTC), but the role of SSTR subtypes in cell growth regulation is still not clear. The present study demonstrates that the human MTC cell line TT stably expresses all the SSTR subtypes and responds to SSTR2 and SSTR5 activation by subtype-selective agonists with two different patterns in terms of [(3)H]thymidine ([(3)H]thy) incorporation and cell number. The SSTR2 preferential agonists (BIM-23120, BIM-23197, BIM-23190, and BIM-23014; 10(-9)-10(-6) M), significantly suppressed [(3)H]thy incorporation (58-13%) and reduced cell proliferation (50-28%), whereas the SSTR5-selective agonist, BIM-23206 (10(-9)-10(-6) M), significantly increased [(3)H]thy incorporation in TT cells (80-175%), but failed to influence cell proliferation. SSTR2 antagonist (BIM-23627) counteracted the action of SSTR2 preferential agonists on TT cells. Furthermore, increasing concentrations of SSTR5-selective agonists, BIM-23206, dose-dependently prevented the suppression of TT cell [(3)H]thy incorporation and proliferation produced by SSTR2 preferential agonist, BIM-23120, showing an antagonism between these compounds. The following conclusions were reached: 1) the human MTC cell line TT expresses all SSTR subtypes; 2) SSTR2 activation inhibits DNA synthesis and cell proliferation, whereas SSTR5 activation increases DNA synthesis; and 3) SSTR2 preferential agonist (BIM-23120) can antagonise SSTR5-selective agonist (BIM-23206) action and vice versa. These findings suggest a tissue-specific function and a tissue-specific interaction between the two receptors.