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Objective: To investigate whether GRK4 regulates the phosphorylation and function of renal CCKBR., Methods: GRK4 A142V transgenic mice were used as an animal model of enhanced GRK4 activity, and siRNA was used to silence the GRK4 gene to investigate the regulatory effect of GRK4 on CCKBR phosphorylation and function. Finally, the co-localization and co-connection of GRK4 and CCKBR in RPT cells were observed by laser confocal microscopy and immunoprecipitation to explore the mechanism of GRK4 regulating CCKBR., Results: Gastrin infusion significantly increased urinary flow and sodium excretion rates in GRK4 WT mice ( P  < .05). GRK4 siRNA did not affect CCKBR protein expression in WKY RPT cells and SHR RPT cells, but remarkably reduced CCKBR phosphorylation in WKY and SHR RPT cells ( P  < .05). The inhibitory effect of gastrin on Na + -K + -ATPase activity in WKY RPT cells was further enhanced by the reduction of GRK4 expression ( P  < .05), while GRK4 siRNA restored the inhibitory effect of gastrin on Na + -K + -ATPase activity in SHR RPT cells. Laser confocal and Co-immunoprecipitation results showed that GRK4 and CCKBR co-localized in cultured RPT cells' cytoplasm., Conclusion: GRK4 participates in the development of hypertension by regulating the phosphorylation of renal CCKBR leading to impaired CCKBR function and water and sodium retention. Knockdown of GRK4 restored the function of CCKBR. The enhanced co-connection between GRK4 and CCKBR may be an important reason for the hyperphosphorylation of GRK4 and CCKBR involved in the pathogenesis of hypertension.

Micro-environmental factors, including stromal and immune cells, cytokines, and circulating hormones are well recognized to determine cancer progression. Melanoma cell growth was recently shown to be suppressed by cholecystokinin/gastrin (CCK) receptor antagonists, and our preliminary data suggested that melanoma patients with Helicobacter gastritis (which is associated with elevated serum gastrin) might have an increased risk of cancer progression. Therefore, in the present study, we examined how gastrin may act on melanoma cells. In 89 melanoma patients, we found a statistically significant association between circulating gastrin concentrations and melanoma thickness and metastasis, which are known risk factors of melanoma progression and prognosis. Immunocytochemistry using a validated antibody confirmed weak to moderate CCK2R expression in both primary malignant melanoma cells and the melanoma cell lines SK-MEL-2 and G361. Furthermore, among the 219 tumors in the Skin Cutaneous Melanoma TCGA Pan-Cancer dataset showing gastrin receptor (CCKBR) expression, significantly higher CCKBR mRNA levels were linked to stage III-IV than stage I-II melanomas. In both cell lines, gastrin increased intracellular calcium levels and stimulated cell migration and invasion through mechanisms inhibited by a CCK2 receptor antagonist. Proteomic studies identified increased MMP-2 and reduced TIMP-3 levels in response to gastrin that were likely to contribute to the increased migration of both cell lines. However, the effects of gastrin on tumor cell invasion were relatively weak in the presence of the extracellular matrix. Nevertheless, dermal fibroblasts/myofibroblasts, known also to express CCK2R, increased gastrin-induced cancer cell invasion. Our data suggest that in a subset of melanoma patients, an elevated serum gastrin concentration is a risk factor for melanoma tumor progression, and that gastrin may act on both melanoma and adjacent stromal cells through CCK2 receptors to promote mechanisms of tumor migration and invasion.

Objective: To observe and explore the effect of Fuling () in alleviating the spleen deficiency symptom pattern (SDSP)., Methods: We established an animal model of SDS in Sprague-Dawley () rats by treating them with deficiency-inducing factors, including irregular feeding and tail clamping. Mice were administered Fuling () and its extracts (raw/cooked powder, aqueous/alcohol extract) by gavage once a day for 21 d. The body weight, rectal temperature, and spleen and thymus organ coefficients were calculated. The levels of motilin (MTL), gastrin (GAS), aquaporin 2 (AQP2), interleukin 2 (IL-2), IL-4, and 5-hydroxytryptamine (5-HT) in the serum and the level of AQP2 in the kidneys were evaluated by enzyme-linked immunosorbent assay., Results: Fuling () and its extracts did not change the body weight, rectal temperature, and organ coefficients of the spleen and thymus. However, it reduced the levels of MTL and GAS and increased the levels of IL-2 and AQP2. In addition, the levels of IL-4 and 5-HT showed no significant alteration., Conclusions: These results suggested the crucial function of () in SDSP, especially promoting digestive function and water metabolism.

Gastrin and CCK (cholecystokinin), gut hormones first secreted after postprandial stages, share the C-terminal amino acids and some types of receptors to be stimulated. Both types of hormone-secreting cells are typical open-type cells which detect foods and their digested elements in the lumen and regulate the secretion of gastric acid and digestive enzymes, gut motility, and satiety. Gastrin cell granules are characterized by their heterogenous ultrastructure within the cell, while CCK cell granules show a uniform ultrastructural figure. Gastrin cells are equipped with peptone receptor GPR92, amino acid receptor GPRC6A, and a Ca-sensing receptor. In addition to nutrient receptors, the release of CCK is regulated by a unique negative feedback mechanism. Development of an antibody for CCK-specific receptor (CCK-1R) has revealed its exact localization throughout the body, but specific antibodies against CCK-2R remain unavailable. Gastrin affects differentiation and proliferation-including cancer cells, while CCK possesses trophic effects to target tissues. CCK is a peripheral satiety signal and acts either via the vagus or directly on the dorsal medulla via CCK-1R. In this review, endocrine cells secreting these unique and so-called old gut hormones are described on a morphological basis.

The present study was designed to observe the effect of quadruple therapy combined with probiotics on Helicobacter pylori -related peptic ulcer. The patients in the control group ( n = 90) were given regular quadruple therapy including proton pump inhibitor ilaprazole enteric-coated tablet + two antibiotics amoxicillin dispersible tablet and metronidazole tablet + colloidal bismuth pectin capsule for 2 weeks. Patients in the study group ( n = 90) were given abovementioned quadruple therapy combined with probiotics live combined Bifidobacterium , Lactobacillus , and Enterococcus Capsules, oral for 2 weeks. Then Hp clearance rate, recurrence rate, levels of gastrointestinal hormone makers, and advance reactions between two groups were compared. At the 2nd week after the treatment, the Helicobacter pylori clearance rate in the study group (87.79%) was significantly higher than the control group (78.89%), and the total recurrence rate in the study group (6.67%) was significantly lower than the control group (13.33%) ( P < 0.05). Serum gastrin and motilin expression were lower, and somatostatin expressions was significantly higher than those in the control group ( P < 0.05). There was no significant difference in the total incidence of adverse reactions between the two groups ( P > 0.05). In summary, quadruple therapy combined with probiotics in the treatment of Helicobacter pylori -related peptic ulcer can improve the Helicobacter pylori clearance rate, reduce the Helicobacter pylori recurrence rate, and is beneficial to improving the level of gastrointestinal hormones, with certain safety., Competing Interests: The authors declare that no conflict of interest is associated with this work., (Copyright © 2022 Ye Zhou and Tingzan Li.)

Background: It is known that increased gastrin concentration is negatively correlated with cardiovascular mortality, and plasma gastrin levels are increased in patients after myocardial infarction (MI). However, whether gastrin can play a protective role in MI remains unknown., Methods: Adult C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery (LAD) and subcutaneous infusion of gastrin (120 μg/Kg body weight/day, 100 μL in the pump) for 28 days after MI. Plasma gastrin concentrations were measured through an ELISA detection kit. Mice were analyzed by echocardiography after surgery. CD31 and VEGF expression were quantified using immunofluorescence staining or/and western blot to assess the angiogenesis in peri-infarct myocardium. Capillary-like tube formation and cell migration assays were performed to detect gastrin-induced angiogenesis., Results: We found that gastrin administration significantly ameliorated MI-induced cardiac dysfunction and reduced fibrosis at 28 days in post-MI hearts. Additionally, gastrin treatment significantly decreased cardiomyocyte apoptosis and increased angiogenesis in the infarct border zone without influencing cardiomyocyte proliferation. In vitro results revealed that gastrin up-regulated the PI3K/Akt/vascular endothelial growth factor (VEGF) signaling pathway and promoted migration and tube formation of human coronary artery endothelial cells (HCAECs). Cholecystokinin 2 receptor (CCK 2 R) mediated the protective effect of gastrin since the CCK 2 R blocker CI988 attenuated the gastrin-mediated angiogenesis and cardiac function protection., Conclusion: Our data revealed that gastrin promoted angiogenesis and improved cardiac function in post-MI mice, highlighting its potential as a therapeutic target candidate., (© 2021. The Author(s).)

Acute myocardial infarction (MI) is one of the leading causes of death in humans. Our previous studies showed that gastrin alleviated acute myocardial ischaemia-reperfusion injury. We hypothesize that gastrin might protect against heart injury after MI by promoting angiogenesis. An MI model was simulated by ligating the anterior descending coronary artery in adult male C57BL/6J mice. Gastrin was administered twice daily by intraperitoneal injection for 2 weeks after MI. We found that gastrin reduced mortality, improved myocardial function with reduced infarct size and promoted angiogenesis. Gastrin increased HIF-1α and VEGF expression. Downregulation of HIF-1α expression by siRNA reduced the proliferation, migration and tube formation of human umbilical vein endothelial cells. These results indicate that gastrin restores cardiac function after MI by promoting angiogenesis via the HIF-1α/VEGF pathway., (© 2021. The Author(s).)

Objectives: To analyse the peptidomics of mouse enteroendocrine cells (EECs) and human gastrointestinal (GI) tissue and identify novel gut derived peptides., Methods: High resolution nano-flow liquid chromatography mass spectrometry (LC-MS/MS) was performed on (i) flow-cytometry purified NeuroD1 positive cells from mouse and homogenised human intestinal biopsies, (ii) supernatants from primary murine intestinal cultures, (iii) intestinal homogenates from mice fed high fat diet. Candidate bioactive peptides were selected on the basis of species conservation, high expression/biosynthesis in EECs and evidence of regulated secretionin vitro. Candidate novel gut-derived peptides were chronically administered to mice to assess effects on food intake and glucose tolerance., Results: A large number of peptide fragments were identified from human and mouse, including known full-length gut hormones and enzymatic degradation products. EEC-specific peptides were largely from vesicular proteins, particularly prohormones, granins and processing enzymes, of which several exhibited regulated secretion in vitro. No regulated peptides were identified from previously unknown genes. High fat feeding particularly affected the distal colon, resulting in reduced peptide levels from GCG, PYY and INSL5. Of the two candidate novel peptides tested in vivo, a peptide from Chromogranin A (ChgA 435-462a) had no measurable effect, but a progastrin-derived peptide (Gast p59-79), modestly improved glucose tolerance in lean mice., Conclusion: LC-MS/MS peptidomic analysis of murine EECs and human GI tissue identified the spectrum of peptides produced by EECs, including a potential novel gut hormone, Gast p59-79, with minor effects on glucose tolerance., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gastric cancer (GC) is one of the most common cancers, with most patients often succumbing to death as a result of tumor metastasis. Recent work has demonstrated that gastrin is closely associated with GC metastasis. However, the specific molecular mechanisms underlying this relationship remain to be unveiled. In this study, we assessed the impact of gastrin and the Wnt/β-catenin inhibitor XAV939 on the epithelial-mesenchymal transition (EMT) of the SGC-7901 and MKN45 GC cell lines, and we determined that gastrin-17 significantly decreased E-cadherin expression and upregulated the expression of Snail1 and N-cadherin in GC cells. In addition, gastrin 17 also significantly increased the expression of Wnt3α in a dose-dependent manner. Consistent with these results, gastrin-17 promoted GC cell invasion, proliferation, and migration in a dose-dependent fashion, and these effects were inhibited by XAV939. Together, these results indicated that gastrin-17 induced GC cell EMT, migration, and invasion via the Wnt/β-catenin signaling pathway, which suggests that this gastrin/Wnt/β-catenin signaling axis may represent a therapeutic target for the prevention of GC metastasis.

Hypertensive nephropathy (HN) is a common cause of end-stage renal disease with renal fibrosis; chronic kidney disease is associated with elevated serum gastrin. However, the relationship between gastrin and renal fibrosis in HN is still unknown. We, now, report that mice with angiotensin II (Ang II)-induced HN had increased renal cholecystokinin receptor B (CCKBR) expression. Knockout of CCKBR in mice aggravated, while long-term subcutaneous infusion of gastrin ameliorated the renal injury and interstitial fibrosis in HN and unilateral ureteral obstruction (UUO). The protective effects of gastrin on renal fibrosis can be independent of its regulation of blood pressure, because in UUO, gastrin decreased renal fibrosis without affecting blood pressure. Gastrin treatment decreased Ang II-induced renal tubule cell apoptosis, reversed Ang II-mediated inhibition of macrophage efferocytosis, and reduced renal inflammation. A screening of the regulatory factors of efferocytosis showed involvement of peroxisome proliferator-activated receptor α (PPAR-α). Knockdown of PPAR-α by shRNA blocked the anti-fibrotic effect of gastrin in vitro in mouse renal proximal tubule cells and macrophages. Immunofluorescence microscopy, Western blotting, luciferase reporter, and Cut&tag-qPCR analyses showed that CCKBR may be a transcription factor of PPAR-α, because gastrin treatment induced CCKBR translocation from cytosol to nucleus, binding to the PPAR-α promoter region, and increasing PPAR-α gene transcription. In conclusion, gastrin protects against HN by normalizing blood pressure, decreasing renal tubule cell apoptosis, and increasing macrophage efferocytosis. Gastrin-mediated CCKBR nuclear translocation may make it act as a transcription factor of PPAR-α, which is a novel signaling pathway. Gastrin may be a new potential drug for HN therapy., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Type 1 diabetes (T1D) results from the autoimmune destruction of β cells, so cure of firmly established T1D requires both reversal of autoimmunity and restoration of β cells. It is known that β cell regeneration in nonautoimmune diabetic mice can come from differentiation of progenitors and/or transdifferentiation of α cells. However, the source of β cell regeneration in autoimmune nonobese diabetic (NOD) mice remains unclear. Here, we show that, after reversal of autoimmunity by induction of haploidentical mixed chimerism, administration of gastrin plus epidermal growth factor augments β cell regeneration and normalizes blood glucose in the firmly established diabetic NOD mice. Using transgenic NOD mice with inducible lineage-tracing markers for insulin-producing β cells, Sox9 + ductal progenitors, Nestin + mesenchymal stem cells, and glucagon-producing α cells, we have found that both reactivation of dysfunctional low-level insulin expression (insulin lo ) β cells and neogenesis contribute to the regeneration, with the latter predominantly coming from transdifferentiation of α cells. These results indicate that, after reversal of autoimmunity, reactivation of β cells and transdifferentiation of α cells can provide sufficient new functional β cells to reach euglycemia in firmly established T1D., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [ 177 Lu]Lu-DOTA-(DGlu) 6 -Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH 2 ([ 177 Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers. Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [ 177 Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation. Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [ 177 Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [ 177 Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [ 177 Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [ 177 Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [ 177 Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging., Competing Interests: Competing Interests: Part of the results of this study have been used for the patent application. M.B. and R.S. are inventors of the patent WO2015/067473: Mini-gastrin analogue, in particular for use in cck2 receptor positive tumor, diagnosis and/or treatment. Debiopharm International SA (Lausanne, Switzerland) and the PSI has a licensing agreement for the clinical development of [177Lu]Lu-PP-F11N. No other potential conflict of interest relevant to this article was reported., (© The author(s).)

MG11 is a truncated analog of minigastrin, a peptide with high affinity and specificity toward the cholecystokinin-2 receptor (CCK2R), which is overexpressed by different tumors. Thus, radiolabeled MG11 derivatives have great potential for use in cancer diagnosis and therapy. A drawback of MG11 is its fast degradation by proteases, leading to moderate tumor uptake in vivo . We introduced 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres to replace labile amide bonds of the peptide. The "triazole scan" yielded peptidomimetics with improved resistance to enzymatic degradation and/or enhanced affinity toward the CCK2R. Remarkably, our lead compound achieved a 10-fold increase in receptor affinity, resulting in a 2.6-fold improved tumor uptake in vivo . Modeling of the ligand-CCK2R complex suggests that an additional cation-π interaction of the aromatic triazole moiety with the Arg 356 residue of the receptor is accountable for these observations. We show for the first time that the amide-to-triazole substitution strategy offers new opportunities in drug development that go beyond the metabolic stabilization of bioactive peptides.

The insertion of single 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres of trans -amide bonds (triazole scan) was recently applied to the 177 Lu-labeled tumor-targeting analog of minigastrin, [Nle 15 ]MG11. The reported novel mono-triazolo-peptidomimetics of [Nle 15 ]MG11 showed either improved resistance against enzymatic degradation or a significantly increased affinity toward the target receptor but never both. To enhance further the tumor-targeting properties of the minigastrin analogs, we studied conjugates with multiple amide-to-triazole substitutions for additive or synergistic effects. Promising candidates were identified by modification of two or three amide bonds, which yielded both improved stability and increased receptor affinity of the peptidomimetics in vitro . Biodistribution studies of radiolabeled multi-triazolo-peptidomimetics in mice bearing receptor-positive tumor xenografts revealed up to 4-fold increased tumor uptake in comparison to the all-amide reference compound [Nle 15 ]MG11. In addition, we report here for the first time a linear peptidomimetic with three triazole insertions in its backbone and maintained biological activity.

The structurally-related peptides, gastrin and cholecystokinin (CCK), were originally discovered as humoral stimulants of gastric acid secretion and pancreatic enzyme release, respectively. With the aid of methodological advances in biochemistry, immunochemistry, and molecular biology in the past several decades, our concept of gastrin and CCK as simple gastrointestinal hormones has changed considerably. Extensive in vitro and in vivo studies have shown that gastrin and CCK play important roles in several cellular processes including maintenance of gastric mucosa and pancreatic islet integrity, neurogenesis, and neoplastic transformation. Indeed, gastrin and CCK, as well as their receptors, are expressed in a variety of tumor cell lines, animal models, and human samples, and might contribute to certain carcinogenesis. In this review, we will briefly introduce the gastrin and CCK system and highlight the effects of gastrin and CCK in the regulation of cell proliferation and apoptosis in both normal and abnormal conditions. The potential imaging and therapeutic use of these peptides and their derivatives are also summarized., (Copyright © 2020 Zeng, Ou, Wang and Guo.)

Gastrin and cholecystokinin (CCK) are hormones released from endocrine cells in the antral stomach (gastrin), the duodenum, and the jejunum (CCK). Recent reports, based on secretion experiments in an enteroendocrine cell line (NCI-H716) and gastrin receptor expression in proglucagon-expressing cells from the rat colon, suggested that gastrin could be a regulator of glucagon-like peptide-1 (GLP-1) secretion. To investigate these findings, we studied the acute effects of CCK-8 (a CCK1/CCK2 (gastrin) receptor agonist) and gastrin-17 (a CCK2(gastrin) receptor agonist) in robust ex vivo models: the isolated perfused rat small intestine and the isolated perfused rat colon. Small intestines from Wistar rats (n = 6), were perfused intraarterially over 80 min. During the perfusion, CCK (1 nmol/L) and gastrin (1 nmol/L) were infused over 10-min periods separated by washout/baseline periods. Colons from Wistar rats (n = 6) were perfused intraarterially over 100 min. During the perfusion, CCK (1 nmol/L), vasoactive intestinal peptide (VIP) (10 nmol/L), and glucose-dependent insulinotropic polypeptide (GIP) (1 nmol/L) were infused over 10-min periods separated by washout/baseline periods. In the perfused rat small intestines neither CCK nor gastrin stimulated the release of GLP-1 or neurotensin. In the perfused rat colon, neither CCK or VIP stimulated GLP-1 or peptide YY (PYY) release, but GIP stimulated both GLP-1 and PYY release. In both sets of experiments, bombesin, a gastrin-releasing peptide analog, served as a positive control. Our findings do not support the suggestion that gastrin or CCK participate in the acute regulation of intestinal GLP-1 secretion, but that GIP may play a role in the regulation of hormone secretion from the colon., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The present study has examined the antidiabetic effects of 21 days co-administration of xenin-8-Gln with the dual-acting fusion peptide, exendin-4/gastrin, as well as persistence of beneficial metabolic benefits, in high fat fed (HFF) mice. Xenin-8-Gln, exendin-4 and gastrin represent compounds that activate receptors of the gut-derived hormones, xenin, glucagon-like peptide-1 (GLP-1) and gastrin, respectively. Twice-daily administration of exendin-4/gastrin, xenin-8-Gln or a combination of both peptides significantly reduced circulating glucose, HbA1c and cumulative energy intake. Combination therapy with xenin-8-Gln and exendin-4/gastrin increased circulating insulin. All HFF mice treated with exendin-4/gastrin presented with body weight similar to lean control mice on day 21. Each treatment improved glucose tolerance and the glucose-lowering actions of glucose dependent insulinotropic polypeptide (GIP), as well as augmenting glucose- and GIP-induced insulin secretion, with benefits being most prominent in the combination group. Administration of exendin-4/gastrin alone, and in combination with xenin-8-Gln, increased pancreatic insulin content and improved the insulin sensitivity index. Pancreatic beta-cell area was significantly increased, and alpha cell area decreased, by all treatments, with the combination group also displaying enhanced overall islet area. Notably, metabolic benefits were generally retained in all groups of HFF mice, and especially in the combination group, following discontinuation of the treatment regimens for 21 days. This was associated with maintenance of increased islet and beta-cell areas. Together, these data confirm the antidiabetic effects of co-activation of GLP-1, gastrin and xenin cell signalling pathways, and highlight the sustainable benefits this type of treatment paradigm can offer in T2DM., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gastrin is a peptide hormone, which in combination with other factors such as TGFα, EGF or GLP-1, is capable of increasing beta cell mass and lowering blood glucose levels in adult diabetic mice. In humans, administration of a bolus of gastrin alone induces insulin secretion suggesting that gastrin may target islet cells. However, whether gastrin alone is sufficient to exert an effect on isolated human islets has been controversial and the mechanism remained poorly understood. Therefore, in this study we started to examine the effects of gastrin alone on cultured adult human islets. Treatment of isolated human islets with gastrin I for 48 h resulted in increased expression of insulin, glucagon and somatostatin transcripts. These increases were significantly correlated with the levels of donor hemoglobin A1c (HbA1c) but not BMI or age. In addition, gastrin treatment resulted in increased expression of PDX1, NKX6.1, NKX2.2, MNX1 and HHEX in islets from donors with HbA1c greater than 42 mmol/mol. The addition of YM022, an antagonist of the gastrin receptor cholecystokinin B receptor (CCKBR), together with gastrin eliminated these effects, verifying that the effects of gastrin are mediated through CCKBR.CCKBR is expressed in somatostatin-expressing delta cells in islets from all donors. However, in the islets from donors with higher HbA1c (greater than 42 mmol/mol [6.0%]), cells triple-positive for CCKBR, somatostatin and insulin were detected, suggesting a de-differentiation or trans-differentiation of endocrine cells. Our results demonstrate a direct effect of gastrin on human islets from prediabetic or diabetic individuals that is mediated through CCKBR+ cells. Further, our data imply that gastrin may be a potential treatment for diabetic patients., Competing Interests: The authors have declared that no competing interests exist.

Angiogenesis is recognized as a sign of cancer and facilitates cancer progression and metastasis. Suppression of angiogenesis is a desirable strategy for gastric cancer (GC) management. In this study, we showed a novel role of gastrin in angiogenesis of GC. We observed that treatment with gastrin 17 (G17) increased the proliferation of AGS cells and enhanced tube formation during normoxia and hypoxia. The expression level of VEGF were increased by G17 treatment as well. Experiments on the mechanism showed that G17 promoted HIF-1α expression, which subsequently enhanced β-catenin nuclear localization and activation of TCF3 and LEF1 and finally resulted in angiogenesis by upregulating VEGF. An in vivo experiment confirmed that G17 enhanced GC cell proliferation and angiogenesis in the resultant tumor. In conclusion, our findings indicate that gastrin promotes angiogenesis via activating HIF-1α/β-catenin/VEGF axis in GC., (Copyright © 2019. Published by Elsevier B.V.)

Derivatized minigastrin analogues make up a promising class of candidates for targeting cholecystokinin receptor subtype 2 (CCK2R), which is overexpressed on cancer cells of various neuroendocrine tumors. The pentaglutamic acid sequence of minigastrin influences its biological properties. In particular, it plays a crucial role in the kidney reuptake mechanism. However, the importance of the binding affinity and interaction of this region with the receptor on a molecular level remains unclear. To elucidate its structure-activity relationship with CCK2R, we replaced this sequence with various linkers differing in their amount of anionic charge, structural characteristics, and flexibility. Specifically, a flexible aliphatic linker, a linker with only three d-Glu residues, and a structured linker with four adjacent β 3 -glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[d-Glu 1-6 ,Nle 11 ]gastrin-13). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the minigastrin derivatives, which allowed radiolabeling with Lutetium-177. The levels of In vitro internalization into MZ-CRC1 cells and in vivo tumor uptake as well as human blood plasma stability increased in the following order: aliphatic linker < three d-Glu < (β 3 -Glu) 4 < (d-Glu) 6 . The in vitro and in vivo behavior was therefore significantly improved with anionic charges. Computational modeling of a CCK2 receptor-ligand complex revealed ionic interactions between cationic residues (Arg and His) of the receptor and anionic residues of the ligand in the linker.

BACKGROUND This study investigated the effect and mechanism of notoginsenoside R1 (NGR1) on chronic atrophic gastritis (CAG) in a rat model. MATERIAL AND METHODS To perform our investigation, a rat model of CAG was established, and then rats were treated with various doses of NGR1. After treatment, hematoxylin-eosin (HE) staining was used for histopathological observation and further scoring. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of gastrointestinal hormones, inflammatory factors, gastric mucosal destruction factors, and gastric mucosal-protective factors. Gene and protein expressions were measured using quantitative real-time PCR and Western blot assay, respectively. RESULTS Results indicated that NGR1 relieved rat CAG. NGR1 treatment significantly increased the levels of gastrin (GAS) and somatostatin (SS) and reduced motilin (MTL) in the serum of CAG rats. The serum levels of interleukin (IL)-1β and IL-6 were significantly reduced by NGR1 treatment in CAG rats in a dose-dependent manner. Additionally, the increased levels of prostaglandin (PG)E2, nitric oxide synthase (NOS), and endothelin (ET) in CAG rats were significantly decreased by NGR1 administration. Moreover, the decreased level of secretory IgA (sIgA) and glutathione (GSH) in rats caused by MNNG was notably increased by NGR1 treatment. No significant changes were found in glutathione disulfide (GSSG) secretion. Finally, we found that the increased Bcl-2 expression and reduced Bax expression in the stomach tissues of rats caused by MNNG were eliminated by NGR1 treatment. CONCLUSIONS NGR1 exerts a protective effect on CAG, and it is a multi-target, multi-linked, comprehensive process.

Background: Ischemia/reperfusion injury (IRI) is one of the most predominant complications of ischemic heart disease. Gastrin has emerged as a regulator of cardiovascular function, playing a key protective role in hypoxia. Serum gastrin levels are increased in patients with myocardial infarction, but the pathophysiogical significance of this finding is unknown. The purpose of this study was to determine whether and how gastrin protects cardiac myocytes from IRI., Methods and Results: Adult male Sprague-Dawley rats were used in the experiments. The hearts in living rats or isolated Langendorff-perfused rat hearts were subjected to ischemia followed by reperfusion to induce myocardial IRI. Gastrin, alone or with an antagonist, was administered before the induction of myocardial IRI. We found that gastrin improved myocardial function and reduced the expression of myocardial injury markers, infarct size, and cardiomyocyte apoptosis induced by IRI. Gastrin increased the phosphorylation levels of ERK1/2 (extracellular signal-regulated kinase 1/2), AKT (protein kinase B), and STAT3 (signal transducer and activator of transcription 3), indicating its ability to activate the RISK (reperfusion injury salvage kinase) and SAFE (survivor activating factor enhancement) pathways. The presence of inhibitors of ERK1/2, AKT, or STAT3 abrogated the gastrin-mediated protection. The protective effect of gastrin was via CCK2R (cholecystokinin 2 receptor) because the CCK2R blocker CI988 prevented the gastrin-mediated protection of the heart with IRI. Moreover, we found a negative correlation between serum levels of cardiac troponin I and gastrin in patients with unstable angina pectoris undergoing percutaneous coronary intervention, suggesting a protective effect of gastrin in human cardiomyocytes., Conclusions: These results indicate that gastrin can reduce myocardial IRI by activation of the RISK and SAFE pathways., (© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Purpose: Metabolic surgery alters the secretion of gastrointestinal hormones that influence glycemic control. Elevated gastrin has been suggested to benefit patients with type 2 diabetes and has been reported following sleeve gastrectomy in rats. The present study compares the effect of hypergastrinemia following sleeve gastrectomy with proton-pump inhibitor therapy on glycemic control and beta-cell mass in lean, diabetic animals., Methods: Thirty-three diabetic Goto-Kakizaki rats were randomized into pantoprazole + sham operation (GK-PPI), sleeve gastrectomy (GK-SG) and vehicle + sham operation (GK-V). Body weight, glucose parameters, HbA1c, glucagon-like peptide 1, gastrin, insulin and lipids were evaluated for eighteen postoperative weeks. Total beta-cell mass was quantified by optical projection tomography., Results: After surgery, body weight development was equal among groups (P g  = 0.75). Fasting and stimulated gastrin increased for GK-PPI and GK-SG vs. GK-V (p < 0.05 for all). Fasting blood glucose was decreased for GK-PPI and GK-SG vs. GK-V (p < 0.05 and p = 0.052). HbA1c was lower for GK-SG vs. GK-V at 6 weeks and for GK-PPI vs. GK-V at twelve- and eighteen weeks postoperative (p < 0.05 for all); a borderline difference was observed for GK-SG vs. GK-V at 18 weeks (p = 0.054). Total- and LDL cholesterol was elevated for GK-PPI compared to the other two groups (p < 0.05 for all). Beta-cell mass did not differ among groups (p = 0.35)., Conclusions: Hypergastrinemia following sleeve gastrectomy and pantoprazole has a similar, modest effect on glycemic control in Goto-Kakizaki rats but does not enhance beta-cell mass after 18 weeks. Hypergastrinemia in the setting of T2DM might be of clinical relevance.

The mechanism by which gastrin promotes pancreatic cancer cell metastasis is unclear. The process of directing polarized cancer cells toward the extracellular matrix is principally required for invasion and distant metastasis; however, whether gastrin can induce this process and its underlying mechanism remain to be elucidated. In this study, we found that gastrin-induced phosphorylation of paxillin at tyrosine 31/118 and RhoA activation as well as promoted the metastasis of PANC-1 cancer cells. Depletion of Gα12 and Gα13 inhibited the phosphorylation of paxillin and downstream activation of GTP-RhoA, blocked the formation and aggregation of focal adhesions and facilitated polarization of actin filaments induced by gastrin. Suppression of RhoA and ROCK also exhibited identical results. Selective inhibition of the CCKBR-Gα12/13-RhoA-ROCK signaling pathway blocked the reoriented localization of the Golgi apparatus at the leading edge of migrated cancer cells. YM022 and Y-27632 significantly suppressed hepatic metastasis of orthotic pancreatic tumors induced by gastrin in vivo. Collectively, we demonstrate that gastrin promotes Golgi reorientation and directional polarization of pancreatic cancer cells by activation of paxillin via the CCKBR-Gα12/13-RhoA-ROCK signal pathway.

Objectives: Modulation of cholecystokinin (CCK) receptors has been shown to influence pancreatic endocrine function., Methods: We assessed the impact of the CCKA and CCKB receptor modulators, (pGlu-Gln)-CCK-8 and gastrin-17, respectively, on β-cell secretory function, proliferation and apoptosis and glucose tolerance, and investigating alterations of CCK and gastrin islet expression in diabetes., Results: Initially, the presence of CCK and gastrin, and expression of their receptors were evidenced in β-cell lines and mouse islets. (pGlu-Gln)-CCK-8 and gastrin-17 stimulated insulin secretion from BRIN-BD11 and 1.1B4 β-cells, associated with no effect on membrane potential or [Ca]i. Only (pGlu-Gln)-CCK-8 possessed insulin secretory actions in isolated islets. In agreement, (pGlu-Gln)-CCK-8 improved glucose disposal and glucose-induced insulin release in mice. In addition, (pGlu-Gln)-CCK-8 evoked clear satiety effects. Interestingly, islet colocalization of CCK with glucagon was elevated in streptozotocin- and hydrocortisone-induced diabetic mice, whereas gastrin coexpression in α cells was reduced. In contrast, gastrin colocalization within β-cells was higher in diabetic mice, while CCK coexpression with insulin was decreased in insulin-deficient mice. (pGlu-Gln)-CCK-8 and gastrin-17 also augmented human and rodent β-cell proliferation and offered protection against streptozotocin-induced β-cell cytotoxicity., Conclusions: We highlight the direct involvement of CCKA and CCKB receptors in pancreatic β-cell function and survival.

Peritoneal metastasis is a major cause of recurrence of gastric cancer and integrins are key molecules involved in gastric cancer cells attachment to the peritoneum. The peptide hormone, gastrin, initially identified for its role in gastric acid secretion is also a growth factor for gastric mucosa. Gastrin has also been shown to contribute to gastric cancers progression. Here, we provide the first evidence that gastrin increases the adhesion of gastric cancer cells. Gastrin treatment induces the expression of α2 integrin subunit through a mechanism that involves the ERK pathway. We also observed in response to gastrin an increase in the amount of α2 integrin associated with β1subunit. In addition, gastrin-stimulated cell adhesion was blocked with an anti-α2β1 integrin neutralizing antibody. We also show that gastrin activates the integrin pathway via the phosphorylation of β1 integrin by a Src family kinase. This mechanism may contribute to the enhancement of cell adhesion observed in response to gastrin since we found an inhibition of gastrin-mediated cell adhesion when cells were treated with a Src inhibitor. By regulating one of the key step of the metastatic process gastrin might contribute to increase the aggressive behaviour of human gastric tumours., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The well-known action of the gastric hormone gastrin in stimulating gastric acid secretion is mediated by activation of cholecystokinin-2 receptors (CCK2R). The latter are expressed by a variety of cell types suggesting that gastrin is implicated in multiple functions. During wound healing in the stomach CCK2R may be expressed by myofibroblasts. We have now characterized CCK2R expression in cultured myofibroblasts. Immunocytochemistry showed that a relatively small proportion (1-6%) of myofibroblasts expressed the receptor regardless of the region of the gut from which they were derived, or whether from cancer or control tissue. Activation of CCK2R by human heptadecapeptide gastrin (hG17) increased intracellular calcium concentrations in a small subset of myofibroblasts indicating the presence of a functional receptor. Unexpectedly, we found over 80% of cells expressing CCK2R were also labeled with 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA during S-phase of the cell cycle. hG17 did not stimulate EdU incorporation but increased migration of both EdU-labeled and unlabelled myofibroblasts; the migratory response was inhibited by a CCK2R antagonist and by an inhibitor of IGF receptor tyrosine kinase; hG17 also increased IGF-2 transcript abundance. The data suggest myofibroblasts express CCK2R in a restricted period of the cell cycle during S-phase, and that gastrin accelerates migration of these cells; it also stimulates migration of adjacent cells probably through paracrine release of IGF. Together with previous findings, the results raise the prospect that gastrin controls the position of dividing myofibroblasts which may be relevant in wound healing and cancer progression in the gastrointestinal tract., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro, colonic organoids cultured from GPR56-/- mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro, and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo, deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

Background: Gastric carcinoids are slow growing neuroendocrine tumours arising from enterochromaffin-like (ECL) cells in the corpus of stomach. Although most of these tumours arise in the setting of gastric atrophy and hypergastrinemia, it is not understood what genetic background predisposes development of these ECL derived tumours. Moreover, diffuse microcarcinoids in the mucosa can lead to a field effect and limit successful endoscopic removal., Objective: To define the genetic background that creates a permissive environment for gastric carcinoids using transgenic mouse lines., Design: The multiple endocrine neoplasia 1 gene locus (Men1) was deleted using Cre recombinase expressed from the Villin promoter (Villin-Cre) and was placed on a somatostatin null genetic background. These transgenic mice received omeprazole-laced chow for 6 months. The direct effect of gastrin and the gastrin receptor antagonist YM022 on expression and phosphorylation of the cyclin inhibitor p27 Kip1 was tested on the human human gastric adenocarcinoma cell line stably expressing CCKBR (AGSE) and mouse small intestinal neuroendocrine carcinoma (STC)-1 cell lines., Results: The combination of conditional Men1 deletion in the absence of somatostatin led to the development of gastric carcinoids within 2 years. Suppression of acid secretion by omeprazole accelerated the timeline of carcinoid development to 6 months in the absence of significant parietal cell atrophy. Carcinoids were associated with hypergastrinemia, and correlated with increased Cckbr expression and nuclear export of p27 Kip1 both in vivo and in gastrin-treated cell lines. Loss of p27 Kip1 was also observed in human gastric carcinoids arising in the setting of atrophic gastritis., Conclusions: Gastric carcinoids require threshold levels of hypergastrinemia, which modulates p27 Kip1 cellular location and stability., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Parietal cells play a fundamental role in stomach maintenance, not only by creating a pathogen-free environment through the production of gastric acid, but also by secreting growth factors important for homeostasis of the gastric epithelium. The gastrointestinal hormone gastrin is known to be a central regulator of both parietal cell function and gastric epithelial cell proliferation and differentiation. Our previous gene expression profiling studies of mouse stomach identified parathyroid hormone-like hormone (PTHLH) as a potential gastrin-regulated gastric growth factor. Although PTHLH is commonly overexpressed in gastric tumors, its normal expression, function, and regulation in the stomach are poorly understood. In this study we used pharmacologic and genetic mouse models as well as human gastric cancer cell lines to determine the cellular localization and regulation of this growth factor by the hormone gastrin. Analysis of Pthlh LacZ/+ knock-in reporter mice localized Pthlh expression to parietal cells in the gastric corpus. Regulation by gastrin was demonstrated by increased Pthlh mRNA abundance after acute gastrin treatment in wild-type mice and reduced expression in gastrin-deficient mice. PTHLH transcripts were also observed in normal human stomach as well as in human gastric cancer cell lines. Gastrin treatment of AGS-E gastric cancer cells induced a rapid and robust increase in numerous PTHLH mRNA isoforms. This induction was largely due to increased transcriptional initiation, although analysis of mRNA half-life showed that gastrin treatment also extended the half-life of PTHLH mRNA, suggesting that gastrin regulates expression by both transcriptional and posttranscriptional mechanisms. NEW & NOTEWORTHY We show that the growth factor parathyroid hormone-like hormone (PTHLH) is expressed in acid-secreting parietal cells of the mouse stomach. We define the specific PTHLH mRNA isoforms expressed in human stomach and in human gastric cancer cell lines and show that gastrin induces PTHLH expression via transcription activation and mRNA stabilization. Our findings suggest that PTHLH is a gastrin-regulated growth factor that might contribute to gastric epithelial cell homeostasis., (Copyright © 2017 the American Physiological Society.)

We previously identified an N-terminal fragment of progastrin in human antrum and plasma, where it circulates in high concentrations. In this study, we examined the effects of N-terminal progastrin fragments on gastric acid secretion by infusion in healthy individuals. Increasing doses of progastrin fragment 1-35 were infused intravenously during constant gastric acid stimulation by gastrin-17. In addition, the effects of progastrin fragment 1-35, fragment 6-35, and fragment 1-19 on gastrin-17 stimulated acid secretion were tested. The gastrin-17 stimulated acid secretion decreased 30% after administration of a high dose of progastrin fragment 1-35 ( P  < 0.05). In extension, a 1-h infusion of fragment 1-35 also decreased gastric acid output. In contrast, fragment 6-35 did not affect acid secretion, and a single infusion of gastrin-17 alone did not reveal fading of gastric acid output during the time course of the experiments. The results show that N-terminal fragments of progastrin may acutely affect gastrin-stimulated gastric acid secretion in vivo. Structure-function analysis suggests that the N-terminal pentapeptide of progastrin is required for the effect., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Objective: The incidence of esophageal adenocarcinoma (EAC) is increasing, but factors contributing to malignant progression of its precursor lesion, Barrett's esophagus (BE), have not been defined. Hypergastrinemia caused by long-term use of proton pump inhibitors (PPIs), has been suggested as one possible risk factor. The gastrin receptor, CCK2R, is expressed in the cardia and upregulated in BE, suggesting the involvement of the gastrin-CCK2R pathway in progression. In the L2-IL-1β mouse model, Barrett's-like esophagus arises from the gastric cardia. Therefore, we aimed to analyze the effect of hypergastrinemia on CCK2R+ progenitor cells in L2-IL-1β mice., Design: L2-IL-1β mice were mated with hypergastrinemic (INS-GAS) mice or treated with PPIs to examine the effect of hypergastrinemia in BE progression. CCK2R-CreERT crossed with L2-IL-1β mice were used to analyze the lineage progenitor potential of CCK2R+ cells. Cardia glands were cultured in vitro, and the effect of gastrin treatment analyzed. L2-IL-1β mice were treated with a CCK2R antagonist YF476 as a potential chemopreventive drug., Results: Hypergastrinemia resulted in increased proliferation and expansion of Barrett's-like esophagus. Lineage tracing experiments revealed that CCK2R+ cells are long-lived progenitors that can give rise to such lesions under chronic inflammation. Gastrin stimulated organoid growth in cardia culture, while CCK2R inhibition prevented Barrett's-like esophagus and dysplasia., Conclusions: Our data suggest a progression model for BE to EAC in which CCK2R+ progenitor cells, stimulated by hypergastrinemia, proliferate to give rise to metaplasia and dysplasia. Hypergastrinemia can result from PPI use, and the effects of hypergastrinemia in human BE should be studied further.

Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension., (Copyright © 2017 the American Physiological Society.)

Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for the diagnosis (imaging) and therapy of cancer. In some cases, the sequences of peptides under investigations contain methionine (Met), an amino acid prone to oxidation during radiolabelling procedures. The formation of oxidative side products can affect the purity of the final radiopharmaceutical product and/or impair its specificity and affinity towards the corresponding receptor. The replacement of Met with oxidation resistant amino acid analogues, for example, norleucine (Nle), can provide a solution. While this approach has been applied successfully to different radiolabelled peptides, a Met → Nle switch only preserves the length of the amino acid side chain important for hydrophobic interactions but not its hydrogen-bonding properties. We report here the use of methoxinine (Mox), a non-canonical amino acid that resembles more closely the electronic properties of Met in comparison to Nle. Specifically, we replaced Met 15 by Mox 15 and Nle 15 in the binding sequence of a radiometal-labelled human gastrin derivative [d-Glu 10 ]HG(10-17), named MG11 (d-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2 ). A comparison of the physicochemical properties of 177 Lu-DOTA[X 15 ]MG11 (X = Met, Nle, Mox) in vitro (cell internalization/externalization properties, receptor affinity (IC 50 ), blood plasma stability and logD) showed that Mox indeed represents a suitable, oxidation-stable amino acid substitute of Met in radiolabelled peptide conjugates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.)

Gastrin, acting via the cholecystokinin-2 receptor (CCK2R), activates its own promoter in a positive-feed-forward loop that may result in hypergastrinemia. Activity of the gastrin promoter is also stimulated by exogenous Zn 2+ ions. Here, the role of intracellular zinc and calcium signaling in the gastrin positive-feed-forward loop was investigated. Gastrin promoter activity was measured in the human gastric carcinoma cell line AGS-CCK2R and in Jurkat cells transfected with various gastrin promoter-luciferase constructs after treatment with gastrin in the presence and absence of zinc- and calcium-chelating agents. The free intracellular zinc ion concentrations were measured in the same cells with the fluorescent indicator FluoZin-3. Cell proliferation and migration/invasion were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell proliferation assay and in Boyden chamber assays, respectively. The zinc chelator N,N,N,N-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) abolished gastrin-stimulated gastrin promoter activity, and the inhibition was completely reversed by exogenous Zn 2+ ions. In contrast, the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) potentiated gastrin-stimulated gastrin promoter activity. Treatment with gastrin increased the intracellular concentration of free Zn 2+ ions, and the increase was blocked by TPEN, but not by BAPTA-AM. TPEN also inhibited the stimulation of cell proliferation and migration/invasion by gastrin, but BAPTA-AM had no effect. These results, which are the first report of the existence of Zn 2+ signaling downstream of CCK2R activation, suggest that zinc chelation therapies may be effective in counteracting gastrin-dependent tumor growth.

Background: Human anion exchanger 1 and 2 (AE1 and AE2) mediate the exchange of Cl(-)/HCO3 (-) across the plasma membrane and regulate intracellular pH (pHi). AE1 is specifically expressed on the surface of erythrocytes, while AE2 is widely expressed in most tissues, and is particularly abundant in parietal cells. Previous studies showed that an interaction between AE1 and p16 is a key event in gastric cancer (GC) progression, but the importance of AE2 in GC is unclear., Methods: The relationship among AE1, AE2 and p16 in GC cells was characterized by molecular and cellular experiments. AE2 expression and pHi were measured after knockdown or forced expression of AE1 or p16 in GC cells. The effect of AE2 on GC growth and the correlation of AE2 expression with differentiation and prognosis of GC were also evaluated. The effect of gastrin on AE2 expression and GC growth was investigated in cellular experiments and mouse xenograft models., Results: p16 binds to both AE1 and AE2 simultaneously. AE1 or p16 silencing elevated AE2 expression on the plasma membrane where it plays a role in pHi regulation and GC suppression. AE2 expression was decreased in GC tissue, and these decreased levels were correlated with poor differentiation and prognosis of GC. The low AE2 protein levels are due to rapid ubiquitin-mediated degradation that was facilitated in the presence of p16. Gastrin inhibited the growth of GC cells at least partially through up-regulation of AE2 expression., Conclusion: AE1/p16 expression promoted AE2 degradation in GC cells. Gastrin is a potential candidate drug for targeted therapies for AE1- and p16-positive GC.

Limitations in β-cell regeneration potential in middle-aged animals could contribute to the increased risk to develop diabetes associated with aging. We investigated β-cell regeneration of middle-aged Wistar rats in response to two different regenerative stimuli: partial pancreatectomy (Px + V) and gastrin administration (Px + G). Pancreatic remnants were analyzed 3 and 14 days after surgery. β-Cell mass increased in young animals after Px and was further increased after gastrin treatment. In contrast, β-cell mass did not change after Px or after gastrin treatment in middle-aged rats. β-Cell replication and individual β-cell size were similarly increased after Px in young and middle-aged animals, and β-cell apoptosis was not modified. Nuclear immunolocalization of neurog3 or nkx6.1 in regenerative duct cells, markers of duct cell plasticity, was increased in young but not in middle-aged Px rats. The pancreatic progenitor-associated transcription factors neurog3 and sox9 were upregulated in islet β-cells of middle-aged rats and further increased after Px. The percentage of chromogranin A+/hormone islet cells was significantly increased in the pancreases of middle-aged Px rats. In summary, the potential for compensatory β-cell hyperplasia and hypertrophy was retained in middle-aged rats, but β-cell dedifferentiation and impaired duct cell plasticity limited β-cell regeneration., (Copyright © 2016 the American Physiological Society.)

Gastrin is absent in most normal adult pancreatic tissues but is highly expressed in pancreatic cancer tissues. Although Gastrin expression was reported to be associated with tumor proliferation in human pancreatic cancer, studies on the relationship between Gastrin and tumor metastasis in pancreatic cancer are rare. In this study, we performed an analysis to determine the effects of Gastrin on modulating the side populations, cell proportion and tumor cell metastatic potential and invasion activity and explored its mechanisms in pancreatic cancer. We indicated that Gastrin and ABCG2 were widely expressed in pancreatic cancer cell lines and overexpressed in cancer tissues. Gastrin induced ABCG2 expression, and this effect was mediated by NF-κB activation. Gastrin regulated the SP proportion of BxPC-3 cells via modulating ABCG2 expression. Through the regulation of the functions of NF-κB/ABCG2, Gastrin functionally promoted the migration and invasion in pancreatic cancer cell. The present study indicated that Gastrin induced ABCG2 expression by activating NF-κB and thereby modulated the SP proportion, tumor cell metastatic potential and invasion activity in pancreatic cancer. Gastrin could serve as an effective therapeutic target for the metastasis of pancreatic cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: In situ inhibition of neutral endopeptidase (NEP) has been recently shown to impressively increase the bioavailability and tumor uptake of biodegradable gastrin radioligands. Furthermore, angiotensin converting enzyme (ACE) has been previously shown to cleave gastrin analogs in vitro. In the present study, we have assessed the effects induced by single or dual NEP/ACE-inhibition on the pharmacokinetic profile of three (99m)Tc-labeled gastrins of varying peptide chain length: [(99m)Tc]SG6 ([(99m)Tc-N4-Gln(1)]gastrin(1-17)), [(99m)Tc]DG2 ([(99m)Tc-N4-Gly(4),DGlu(5)]gastrin(4-17)) and [(99m)Tc]DG4 ([(99m)Tc-N4-DGlu(10)]gastrin(10-17))., Methods: Mouse blood samples were collected 5min after injection of each of [(99m)Tc]SG6/DG2/DG4 together with: a) vehicle, b) the NEP-inhibitor phosphoramidon (PA), c) the ACE-inhibitor lisinopril (Lis), or d) PA plus Lis and were analyzed by RP-HPLC for radiometabolite detection. Biodistribution was studied in SCID mice bearing A431-CCK2R(+/-) xenografts at 4h postinjection (pi). [(99m)Tc]SG6 or [(99m)Tc]DG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for [(99m)Tc]DG2 control and PA animal groups were only included., Results: Treatment of mice with PA induced significant stabilization of (99m)Tc-radiotracers in peripheral blood, while treatment with Lis or Lis+PA affected the stability of des(Glu)5 [(99m)Tc]DG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (P<0.01). Only [(99m)Tc]DG4 profited by single Lis (2.06±0.39%ID/g vs 0.99±0.13%ID/g in controls) or combined Lis+PA coinjection (8.91±1.61%ID/g vs 4.89±1.33%ID/g in PA-group). Furthermore, kidney uptake remained favourably low and unaffected by PA and/or Lis coinjection only in the case of [(99m)Tc]DG4 (<1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios., Conclusions: In situ NEP/ACE-inhibition diversely affected the in vivo profile of (99m)Tc-radioligands based on different-length gastrins. Truncated [(99m)Tc]DG4 exhibited overall the most attractive profile during combined NEP/ACE-inhibition in mouse models, providing new opportunities for CCK2R-expressing tumor imaging in man with SPECT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Intestinal ischemia-reperfusion (I/R) injury is a devastating complication when the blood supply is reflowed in ischemic organs. Gastrin has critical function in regulating acid secretion, proliferation, and differentiation in the gastric mucosa. We aimed to determine whether gastrin has an effect on intestinal I/R damage. Intestinal I/R injury was induced by 60-min occlusion of the superior mesenteric artery followed by 60-min reperfusion, and the rats were induced to be hypergastrinemic by pretreated with omeprazole or directly injected with gastrin. Some hypergastrinemic rats were injected with cholecystokinin-2 (CCK-2) receptor antagonist prior to I/R operation. After the animal surgery, the intestine was collected for histological analysis. Isolated intestinal epithelial cells or crypts were harvested for RNA and protein analysis. CCK-2 receptor expression, intestinal mucosal damage, cell apoptosis, and apoptotic protein caspase-3 activity were measured. We found that high gastrin in serum significantly reduced intestinal hemorrhage, alleviated extensive epithelial disruption, decreased disintegration of lamina propria, downregulated myeloperoxidase activity, tumor necrosis factor-α, and caspase-3 activity, and lead to low mortality in response to I/R injury. On the contrary, CCK-2 receptor antagonist L365260 could markedly impair intestinal protection by gastrin on intestinal I/R. Severe edema of mucosal villi with severe intestinal crypt injury and numerous intestinal villi disintegrated were observed again in the hypergastrinemic rats with L365260. The survival in the hypergastrinemic rats after intestinal I/R injury was shortened by L365260. Finally, gastrin could remarkably upregulated intestinal CCK-2 receptor expression. Our data suggest that gastrin by omeprazole remarkably attenuated I/R induced intestinal injury by enhancing CCK-2 receptor expression and gastrin could be a potential mitigator for intestinal I/R damage in the clinical setting., (© 2016 by the Society for Experimental Biology and Medicine.)

Background: Gastrokine 1 (GKN1) acts as a gastric tumor suppressor. Here, we investigated whether GKN1 contributes to the maintenance of gastric mucosal homeostasis by regulating gastrin-induced gastric epithelial cell growth., Methods: We assessed the effects of gastrin and GKN1 on cell proliferation in stable AGS(GKN1) and MKN1(GKN1) gastric cancer cell lines and HFE-145 nonneoplastic epithelial cells. Cell viability and proliferation were analyzed by MTT and BrdU incorporation assays, respectively. Cell cycle and expression of growth factor receptors were examined by flow cytometry and Western blot analyses., Results: Gastrin treatment stimulated a significant time-dependent increase in cell viability and proliferation in AGS(mock) and MKN1(mock), but not in HFE-145, AGS(GKN1), and MKN1(GKN1), cells, which stably expressed GKN1. Additionally, gastrin markedly increased the S-phase cell population, whereas GKN1 significantly inhibited the effect of gastrin by regulating the expression of G1/S cell-cycle regulators. Furthermore, gastrin induced activation of the NF-kB and β-catenin signaling pathways and increased the expression of CCKBR, EGFR, and c-Met in AGS and MKN1 cells. However, GKN1 completely suppressed these effects of gastrin via downregulation of gastrin/CCKBR/growth factor receptor expression. Moreover, GKN1 reduced gastrin and CCKBR mRNA expression in AGS and MKN1 cells, and there was an inverse correlation between GKN1 and gastrin, as well as between GKN1 and CCKBR mRNA expression in noncancerous gastric mucosae., Conclusion: These data suggest that GKN1 may contribute to the maintenance of gastric epithelial homeostasis and inhibit gastric carcinogenesis by downregulating the gastrin-CCKBR signaling pathway.

The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na(+),K(+)-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na(+),K(+)-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na(+),K(+)-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na(+),K(+)-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes., (Copyright © 2016 the American Physiological Society.)

We previously reported that long-term administration of a low dose of gastrin and epidermal growth factor (GE) augments β-cell neogenesis in late-stage diabetic autoimmune mice after eliminating insulitis by induction of mixed chimerism. However, the source of β-cell neogenesis is still unknown. SRY (sex-determining region Y)-box 9(+) (Sox9(+)) ductal cells in the adult pancreas are clonogenic and can give rise to insulin-producing β cells in an in vitro culture. Whether Sox9(+) ductal cells in the adult pancreas can give rise to β cells in vivo remains controversial. Here, using lineage-tracing with genetic labeling of Insulin- or Sox9-expressing cells, we show that hyperglycemia (>300 mg/dL) is required for inducing Sox9(+) ductal cell differentiation into insulin-producing β cells, and medium hyperglycemia (300-450 mg/dL) in combination with long-term administration of low-dose GE synergistically augments differentiation and is associated with normalization of blood glucose in nonautoimmune diabetic C57BL/6 mice. Short-term administration of high-dose GE cannot augment differentiation, although it can augment preexisting β-cell replication. These results indicate that medium hyperglycemia combined with long-term administration of low-dose GE represents one way to induce Sox9(+) ductal cell differentiation into β cells in adult mice.

Non-amidated gastrin peptides such as glycine-extended gastrin (Ggly) are biologically active in vitro and in vivo and have been implicated in the development of gastric and colonic cancers. Previous studies have shown that the truncated form of Ggly, the octapeptide LE5AY, was still biologically active in vitro, and that activity was dependent on ferric ion binding but independent of binding to the cholecystokinin 2 (CCK2) receptor. The present work was aimed at creating more stable gastrin-derived 'super agonists' using retro-inverso technology. The truncated LE5AY peptide was synthesized using end protecting groups in three forms with l-amino acids (GL), d-amino acids (GD) or retro-inverso (reverse order with d-amino acids; GRI). All of these peptides bound ferric ions with a 2:1 (Fe: peptide) ratio. As predicted, Ggly, GL and GRI were biologically active in vitro and increased cell proliferation in mouse gastric epithelial (IMGE-5) and human colorectal cancer (DLD-1) cell lines, and increased cell migration in DLD-1 cells. These activities were likely via the same mechanism as Ggly since no CCK1 or CCK2 binding was identified, and GD remained inactive in all assays. Surprisingly, unlike Ggly, GL and GRI were not active in vivo. While Ggly stimulated colonic crypt height and proliferation rates in gastrin knockout mice, GL and GRI did not. The apparent lack of activity may be due to rapid clearance of these smaller peptides. Nevertheless further work designing and testing retro-inverso gastrins is warranted, as it may lead to the generation of super agonists that could potentially be used to treat patients with gastrointestinal disorders with reduced mucosal function., (Copyright © 2015 Elsevier Inc. All rights reserved.)

One week of treatment with EGF and gastrin (EGF/G) was shown to restore normoglycemia and to induce islet regeneration in mice treated with the diabetogenic agent alloxan. The mechanisms underlying this regeneration are not fully understood. We performed genetic lineage tracing experiments to evaluate the contribution of beta cell neogenesis in this model. One day after alloxan administration, mice received EGF/G treatment for one week. The treatment could not prevent the initial alloxan-induced beta cell mass destruction, however it did reverse glycemia to control levels within one day, suggesting improved peripheral glucose uptake. In vitro experiments with C2C12 cell line showed that EGF could stimulate glucose uptake with an efficacy comparable to that of insulin. Subsequently, EGF/G treatment stimulated a 3-fold increase in beta cell mass, which was partially driven by neogenesis and beta cell proliferation as assessed by beta cell lineage tracing and BrdU-labeling experiments, respectively. Acinar cell lineage tracing failed to show an important contribution of acinar cells to the newly formed beta cells. No appearance of transitional cells co-expressing insulin and glucagon, a hallmark for alpha-to-beta cell conversion, was found, suggesting that alpha cells did not significantly contribute to the regeneration. An important fraction of the beta cells significantly lost insulin positivity after alloxan administration, which was restored to normal after one week of EGF/G treatment. Alloxan-only mice showed more pronounced beta cell neogenesis and proliferation, even though beta cell mass remained significantly depleted, suggesting ongoing beta cell death in that group. After one week, macrophage infiltration was significantly reduced in EGF/G-treated group compared to the alloxan-only group. Our results suggest that EGF/G-induced beta cell regeneration in alloxan-diabetic mice is driven by beta cell neogenesis, proliferation and recovery of insulin. The glucose-lowering effect of the treatment might play an important role in the regeneration process.

Cholecystokinin (CCK) receptors are G-protein coupled receptors (GPCR) which are present on lung cancer cells. CCK-8 stimulates the proliferation of lung cancer cells, whereas the CCK2R receptor antagonist CI-988 inhibits proliferation. GPCR for some gastrointestinal hormones/neurotransmitters mediate lung cancer growth by causing epidermal growth factor receptor (EGFR) transactivation. Here, the role of CCK/gastrin and CI-988 on EGFR transactivation and lung cancer proliferation was investigated. Addition of CCK-8 or gastrin-17 (100 nM) to NCI-H727 human lung cancer cells increased EGFR Tyr(1068) phosphorylation after 2 min. The ability of CCK-8 to cause EGFR tyrosine phosphorylation was blocked by CI-988, gefitinib (EGFR tyrosine kinase inhibitor), PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), and tiron (superoxide scavenger). CCK-8 nonsulfated and gastrin-17 caused EGFR transactivation and bound with high affinity to NCI-H727 cells, suggesting that the CCK2R is present. CI-988 inhibited the ability of CCK-8 to cause ERK phosphorylation and elevate cytosolic Ca(2+). CI-988 or gefitinib inhibited the basal growth of NCI-H727 cells or that stimulated by CCK-8. The results indicate that CCK/gastrin may increase lung cancer proliferation in an EGFR-dependent manner.

Angiogenesis is essential in tumor progression and metastatic process, and increased angiogenesis has been associated with poor prognosis and relapse of colorectal cancer (CRC). VEGF has become the main target of anti-angiogenic therapy. However, most patients relapse after an initial response or present a resistance to the treatment. Identification of new pro-angiogenic factors may help to improve anti-angiogenic therapy. In this study, we demonstrated that the pro-hormone progastrin (PG), over-expressed in CRC, recognized as a growth factor, is a potent pro-angiogenic factor. In transgenic mice and human colorectal HPs producing high levels of PG, we correlated PG overexpression with an increased vascularization. In vitro, exogenous PG and conditioned media (CM) from CRC cells producing PG increased endothelial cell proliferation and migration. We also showed that treatment with exogenous PG can increase the ability of endothelial cells to form capillary-like structures. Moreover, we demonstrated that PG enhanced endothelial permeability. The finding that PG stimulated the phosphorylation of vascular endothelial (VE)-cadherin, p125-FAK, paxillin and induced actin remodelling was consistent with a role of these components in PG-stimulated endothelial cell migration and permeability. The pro-angiogenic effects observed with CM were significantly inhibited when CRC cells expressed a PG shRNA. In vivo, we found an important decrease in tumor growth and neovascularization when the CRC cells expressing the PG shRNA were xenografted in mice or in the chick chorioallantoic membrane model. We also observed an increase in the coverage of blood vessels by pericytes and a decrease in endothelial permeability when PG expression was blocked. Our results demonstrate that PG is a new pro-angiogenic factor in CRC and an attractive therapeutic target.

Gastrin is a gastrointestinal hormone secreted by G cells. Hypergastrinemia can improve blood glucose and glycosylated hemoglobin levels. These positive effects are primarily due to the trophic effects of gastrin on β-cells. In recent years, many receptors that regulate secretion of glucagon-like peptide 1 (GLP-1) have been identified in enteroendocrine L cell lines. This led us to hypothesize that, in addition to the trophic effects of gastrin on β-cells, L cells also express cholecystokinin2-receptor (CCK2R), which may regulate GLP-1 secretion and have synergistic effects on glucose homeostasis. Our research provides a preliminary analysis of CCK2R expression and the stimulating effect of gastrin treatment on GLP-1 secretion in a human endocrine L cell line, using RT-PCR, Western blot, immunocytochemistry, and ELISA analyses. The expression of proglucagon and prohormone convertase 3, which regulate GLP-1 biosynthesis, were also analyzed by real-time PCR. Double immunofluorescence labeling was utilized to assess the intracellular localization of CCK2R and GLP-1 in L cells harvested from rat colon tissue. Our results showed that CCK2R was expressed in both the human L cell line and the rat L cells. We also showed that treatment with gastrin, a CCK2R agonist, stimulated the secretion of GLP-1, and that this effect was likely due to increased expression of proglucagon and PCSK1 (also known as prohormone convertase 3 (PC3 gene)). These results not only provide a basis for the role gastrin may play in intestinal L cells, and may also provide the basis for the development of a method of gastrin-mediated glycemic regulation., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: Cholecystokinin receptor subtype 2 (CCK-2) is overexpressed in various tumours like medullary thyroid carcinomas and small cell lung cancer. Radiolabelled peptides that bind with high affinity and specificity to CCK-2 receptors, thus hold great potential for visualizing such tumours., Methods: We compared four 111In labelled gastrin analogues, called minigastrins (MG), namely MG11, MG45, MG47 and MG48 linked to metal chelating DOTA in preclinical experiments. The radiolabelled peptides were tested for peptide binding in CCK-2 receptor-bearing cell line AR42J and for their pharmacokinetics in normal rats., Results: The experiments suggest that all gastrin analogues had similar and relatively rapid internalization into AR42J cells. Binding to CCK-2 receptors in AR42J cells was saturable for all agents but there were some differences in receptor affinity. This biodistribution study in rats showed a rapid decrease in blood radioactivity, predominantly renal clearance and saturable uptake of the radiopharmaceutical and/or its metabolites in the CCK-2 receptor-positive stomach. Higher kidney accumulation of radioactivity was only found for 111In-DOTA-minigastrin 48., Conclusions: The data suggest that the 111In-DOTA-minigastrin analogues studied are promising candidates for the scintigraphy of CCK-2 receptor-expressing tumours; 111In-DOTA-MG47 and 111In-DOTA-MG11 are the most promising.