Cholecystokinin Analogue Research Articles

Novel Pharmaceuticals in Appetite Regulation: Exploring emerging gut peptides and their pharmacological prospects.

Obesity, a global health challenge, necessitates innovative approaches for effective management. Targeting gut peptides in the development of anti-obesity pharmaceuticals has already demonstrated significant efficacy. Ghrelin, peptide YY (PYY), cholecystokinin (CCK), and amylin are crucial in appetite regulation offering promising targets for pharmacological interventions in obesity treatment using both peptide-based and small molecule-based pharmaceuticals. Ghrelin, a sole orexigenic gut peptide, has a potential for anti-obesity therapies through various approaches, including endogenous ghrelin neutralization, ghrelin receptor antagonists, ghrelin O-acyltransferase, and functional inhibitors. Anorexigenic gut peptides, peptide YY, cholecystokinin, and amylin, have exhibited appetite-reducing effects in animal models and humans. Overcoming substantial obstacles is imperative for translating these findings into clinically effective pharmaceuticals. Peptide YY and cholecystokinin analogues, characterized by prolonged half-life and resistance to proteolytic enzymes, present viable options. Positive allosteric modulators emerge as a novel approach for modulating the cholecystokinin pathway. Amylin is currently the most promising, with both amylin analogues and dual amylin and calcitonin receptor agonists (DACRAs) progressing to advanced stages of clinical trials. Despite persistent challenges, innovative pharmaceutical strategies provide a glimpse into the future of anti-obesity therapies.

Identification of sulfakinin receptor regulating feeding behavior and hemolymph trehalose homeostasis in the silkworm, Bombyx mori

Feeding behavior, the most fundamental physiological activity, is controlled by two opposing groups of factors, orexigenic and anorexigenic factors. The sulfakinin family, an insect analogue of the mammalian satiety factor cholecystokinin (CCK), has been shown to suppress food intake in various insects. Nevertheless, the mechanisms through which sulfakinin regulates feeding behavior remain a biological question. This study aimed to elucidate the signaling pathway mediated by the anorexigenic peptide sulfakinin in Bombyx mori. We identified the Bombyx mori neuropeptide G protein-coupled receptor A9 (BNGR-A9) as the receptor for sulfakinin through functional assays. Stimulation with sulfakinin triggered a swift increase in intracellular IP3, Ca2+, and a notable enhancement of ERK1/2 phosphorylation, in a manner sensitive to a Gαq-specific inhibitor. Treatment with synthetic sulfakinin resulted in decreased food consumption and average body weight. Additionally, administering synthetic sulfakinin to silkworms significantly elevated hemolymph trehalose levels, an effect markedly reduced by pre-treatment with BNGR-A9 dsRNA. Consequently, our findings establish the sulfakinin/BNGR-A9 signaling pathway as a critical regulator of feeding behavior and hemolymph trehalose homeostasis in Bombyx mori, highlighting its roles in the negative control of food intake and the positive regulation of energy balance.

Exploring FDA-Approved Frontiers: Insights into Natural and Engineered Peptide Analogues in the GLP-1, GIP, GHRH, CCK, ACTH, and α-MSH Realms.

Peptides continue to gain significance in the pharmaceutical arena. Since the unveiling of insulin in 1921, the Food and Drug Administration (FDA) has authorised around 100 peptides for various applications. Peptides, although initially derived from endogenous sources, have evolved beyond their natural origins, exhibiting favourable therapeutic effectiveness. Medicinal chemistry has played a pivotal role in synthesising valuable natural peptide analogues, providing synthetic alternatives with therapeutic potential. Furthermore, key chemical modifications have enhanced the stability of peptides and strengthened their interactions with therapeutic targets. For instance, selective modifications have extended their half-life and lessened the frequency of their administration while maintaining the desired therapeutic action. In this review, I analyse the FDA approval of natural peptides, as well as engineered peptides for diabetes treatment, growth-hormone-releasing hormone (GHRH), cholecystokinin (CCK), adrenocorticotropic hormone (ACTH), and α-melanocyte stimulating hormone (α-MSH) peptide analogues. Attention will be paid to the structure, mode of action, developmental journey, FDA authorisation, and the adverse effects of these peptides.

Use of a Fatty Meal Cholecystagogue Protocol in Hepatobiliary Scintigraphy for Chronic Functional Gallbladder Disease.

Chronic functional gallbladder disorder, characterized by biliary pain in the absence of structural pathology, poses a diagnostic challenge necessitating reliable cholecystagogues for accurate evaluation. However, recurrent shortages of synthetic cholecystokinin analogs have prompted the exploration of alternative agents. This paper describes the efficacy of Ensure Plus as a viable fatty meal substitute for hepatobiliary scintigraphy in assessing chronic functional gallbladder disorder. Through comparative studies, Ensure Plus demonstrates comparable diagnostic accuracy to cholecystokinin in similar patient populations. Furthermore, Ensure Plus demonstrates significant symptom improvement after cholecystectomy in patients with anomalous gallbladder ejection fractions. This paper offers a detailed protocol for the seamless integration of Ensure Plus into hepatobiliary scintigraphy, providing clinicians with a valuable tool to navigate cholecystokinin shortages while maintaining diagnostic precision in cases of chronic functional gallbladder disorder. The use of Ensure Plus not only addresses practical supply challenges but also underscores its potential as a cost-effective and clinically sound alternative in biliary diagnostics.

α‑lipoic acid inhibits cerulein/resistin‑induced expression of interleukin‑6 by activating peroxisome proliferator‑activated receptor‑γ in pancreatic acinar cells.

Cerulein-induced pancreatitis resembles human acute pancreatitis in terms of pathological events, such as enzymatic activation and inflammatory cell infiltration in the pancreas. Cerulein is a cholecystokinin analog that increases levels of reactive oxygen species (ROS) and interleukin-6 (IL-6) expression level in pancreatic acinar cells. Serum levels of resistin, which is secreted from adipocytes, are reportedly higher in patients with acute pancreatitis than in healthy individuals. Previously, it was shown that the adipokine resistin can aggravate the cerulein-induced increase in ROS levels and IL-6 expression level in pancreatic acinar cells. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) is a key regulator of the transcription and expression of antioxidant enzymes, including heme oxygenase 1 (HO-1) and catalase. α-lipoic acid, a naturally occurring dithiol antioxidant, can prevent cerulein-induced pancreatic damage in rats. In the present study, it was aimed to investigate whether α-lipoic acid can attenuate the cerulein/resistin-induced increase in IL-6 expression and ROS levels via PPAR-γ activation in pancreatic acinar AR42J cells. The anti-inflammatory mechanism of α-lipoic acid was determined using reverse transcription-quantitative PCR, western blot analysis, enzyme-linked immunosorbent assay, immunofluoresc2ence staining and fluorometry. Treatment with cerulein and resistin increased ROS levels and IL-6 expression level, which were inhibited by α-lipoic acid in pancreatic acinar cells. α-lipoic acid increased the nuclear translocation and expression level of PPAR-γ and the expression levels of its target genes: HO-1 and catalase. The PPAR-γ antagonist GW9662 and HO-1 inhibitor zinc protoporphyrin reversed the inhibitory effect of α-lipoic acid on cerulein/resistin-induced increase in ROS and IL-6 levels. In conclusion, α-lipoic acid inhibits the cerulein/resistin-induced increase in ROS production and IL-6 expression levels by activating PPAR-γ and inducing the expression of HO-1 and catalase in pancreatic acinar cells.

Tricetin Reduces Inflammation and Acinar Cell Injury in Cerulein-Induced Acute Pancreatitis: The Role of Oxidative Stress-Induced DNA Damage Signaling.

Acute pancreatitis (AP) poses a worldwide challenge due to the growing incidence and its potentially life-threatening course and complications. Specific targeted therapies are not available, prompting the identification of new pathways and novel therapeutic approaches. Flavonoids comprise several groups of biologically active compounds with wide-ranging effects. The flavone compound, tricetin (TCT), has not yet been investigated in detail but sporadic reports indicate diverse biological activities. In the current study, we evaluated the potential protective effects of TCT in AP. TCT (30 μM) protected isolated primary murine acinar cells from the cytotoxic effects of cerulein, a cholecystokinin analog peptide. The protective effects of TCT were observed in a general viability assay (calcein ester hydrolysis), in an apoptosis assay (caspase activity), and in necrosis assays (propidium iodide uptake and lactate dehydrogenase release). The effects of TCT were not related to its potential antioxidant effects, as TCT did not protect against H2O2-induced acinar cell death despite possessing radical scavenging activity. Cerulein-induced expression of IL1β, IL6, and matrix metalloproteinase 2 and activation of nuclear factor-κB (NFκB) were reduced by 30 μM TCT. In vivo experiments confirmed the protective effect of TCT in a mouse model of cerulein-induced AP. TCT suppressed edema formation and apoptosis in the pancreas and reduced lipase and amylase levels in the serum. Moreover, TCT inhibited interleukin-1β (IL1β), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) expression in the pancreas and reduced the activation of the oxidative DNA damage sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Our data indicate that TCT can be a potential treatment option for AP.

Cholecystokinin and glucagon-like peptide-1 analogues regulate intestinal tight junction, inflammation, dopaminergic neurons and α-synuclein accumulation in the colon of two Parkinson's disease mouse models

Parkinson's disease (PD) is the second most common neurodegenerative disease, and no treatment is available to stop its progression. Studies have shown that the colonic pathology of PD precedes that of the brain. The 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and the human A53T α-synuclein (α-syn) transgenic PD mouse model show colonic pathology and intestinal dopaminergic neuronal damage, which is comparable to the intestinal pathology of PD. Cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1), which are brain-gut peptides, have neurotrophic and anti-inflammatory properties. Two GLP-1R agonists have already shown robust effects in phase II trials in PD patients. However, whether they have beneficial effects on colonic pathology in PD remains unclear. In this study, MPTP-treated mice and human A53T α-syn transgenic mice were intraperitoneally injected with a CCK analogue or Liraglutide, a GLP-1 analogue, once a day for 5 weeks. Levels of colonic epithelial tight junction proteins including occludin and zonula occludens-1 (ZO-1), inflammatory biomarkers including inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α), brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH) and α-syn were analyzed. The results show that the CCK analogue and Liraglutide both restored the disruption of intestinal tight junction, reduced colonic inflammation, inhibited colonic dopaminergic neurons reduction and the accumulation of α-syn oligomers in the colon of both PD mice models. This study suggested that CCK or GLP-1 analogues could be beneficial to the improvement of leaky gut barrier, inflammation, dopaminergic neuron impairment and accumulation of α-syn in the colon of PD patients.

Activation of dorsal motor nucleus cholinergic neurons ameliorates the severity of acute pancreatitis

Clusters of neuron cell bodies, termed nuclei, residing in the brainstem are the origin of cranial nerves which transmit action potentials controlling organ function. Neurons residing in the brainstem dorsal motor nucleus (DMN) project in the vagus nerve to communicate with the pancreas, liver, gastrointestinal tract and other organs. Cholinergic signaling in the vagus nerve‐mediated inflammatory reflex also controls immune system responses by inhibiting the production of cytokines in the spleen, although the function of DMN neurons in regulating pancreatic inflammation is not known. Here, we demonstrate that selective activation of cholinergic neurons in the DMN attenuates pancreatitis severity. Pancreatitis was induced with two intraperitoneal injections of the cholecystokinin analogue caerulein (50 mcg/kg), given one hour apart. A fiber‐optic cannula was inserted under stereotactic guidance into the DMN in transgenic mice expressing light‐responsive ion channel, channelrhodopsin, in cholinergic neurons under the choline acetyltransferase promoter. Animals were subjected to either optogenetic stimulation (473nm, 20 Hz, 25% duty cycle, 8‐12 mW total power) or sham stimulation (no light) for five minutes. Optogenetic stimulation but not sham stimulation of the cholinergic neurons in the DMN significantly attenuates serum amylase (4043mU/mL ± 264.4 vs. 2940mU/mL ± 170.9, p=0.0074; n=9‐11 mice/group) and pancreatic IL‐6 levels (1907pg/mg ± 879.3 vs. 1046pg/mg± 288.1, p=0.0330; n=9‐11 mice/group). The decreased severity of pancreatitis in optogenetically stimulated mice is further demonstrated by a significant improvement in pancreatic histologic severity score (6.86 ± 1.67 vs. 5.35 ± 1.44, p = 0.0462; n = 9‐11). Pharmacological blockade and surgical ablation of the vagus nerve signaling inhibit protective effects of DMN cholinergic neurons activation. Pre‐treatment with mecamylamine, a nicotinic receptor antagonist, attenuates the reduction in serum amylase following light stimulation (Sham stimulation vs. DMN vs DMN + Mecamylamine: 5047 ± 315.4 vs. 3769 ± 241.5 vs. 5620 ± 385.9, p = 0037; n = 10 mice/group). Additionally, mice with subdiaphragmatic vagotomy has no difference in serum amylase levels following optogenetic stimulation (2761mU/mL ± 787.3 vs. 2517mU/mL, p= 0.300; n=3), whereas sham surgery controls show a significant reduction in serum amylase levels (3224mU/mL ± 593.1 vs. 2087 mU/mL ± 951.2, p=0.0303; n=6 mice/group). These studies identify DMN as an important locus that regulates the severity of acute pancreatitis in vagus‐nerve‐dependent fashion, and provide new insights into the identity and central origin of the efferent vagus nerve fibers regulating acute pancreatitis.

A Mouse Model of Acute and Chronic Pancreatitis.

Pancreatitis occurs in two forms defined by its chronicity. Acute pancreatitis (AP) occurs suddenly and only lasts for several days. Consequently, most patients with AP recover without permanent damage to the pancreas, and about 20% of patients with AP have severe disease. In contrast, chronic pancreatitis (CP) is a long-lasting inflammation that causes permanent damage to pancreatic tissue; consequently, this form is marked by the emergence of persistent endocrine and exocrine pancreatic insufficiency. Despite these differences, AP and CP share central mechanisms of disease: in both forms, inflammation is initiated and/or sustained by the intrapancreatic activation of pancreatic digestive enzymes followed by the autodigestion of pancreatic tissues. In addition, in both forms enzymatic damage is accompanied by changes in intestinal permeability and entry of commensal organisms into the pancreas where they elicit innate immune responses that ultimately dominate and define pancreatic inflammation. In the murine models of AP and CP described here, both of these elements of pancreatitis pathogenesis are taken into account. Thus, in one approach mice are administered high doses of cerulein, a cholecystokinin analog with the ability at this dose to induce excessive activation of the cholecystokinin receptor expressed in pancreatic acinar cells and the release of active trypsin that causes both direct and indirect acinar damages due to entry of commensal organisms and stimulation of innate immune responses. In a second approach mice are administered low doses of cerulein, which causes little or no damage to the pancreas unless given along with nucleotide-binding oligomerization domain 1 (NOD1) ligand, which in the presence of low-dose cerulein administration induces a pathologic innate immune response mediated by NOD1. These approaches are adopted to produce AP when cerulein or cerulein plus NOD1 ligand is applied only once or to produce CP when a similar regimen is applied multiple times. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cerulein-induced acute pancreatitis Alternate Protocol 1: Acute pancreatitis induced by cerulein and NOD1 ligand Basic Protocol 2: Cerulein-induced chronic pancreatitis Alternate Protocol 2: Chronic pancreatitis induced by cerulein and NOD1 ligand Support Protocol: Isolation of pancreatic mononuclear cells.

Neuroprotective Effects of a Cholecystokinin Analogue in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Parkinson's Disease Mouse Model.

Parkinson’s disease (PD) is a chronic neurodegenerative disease. Type 2 diabetes mellitus (T2DM) has been identified as a risk factor for PD. Drugs originally developed for T2DM treatment such as liraglutide have shown neuroprotective effects in mouse models of PD. Cholecystokinin (CCK) is a peptide hormone with growth factor properties. Here, we demonstrate the neuroprotective effects of the (pGLu)-(Gln)-CCK8 analogue in an acute PD mouse model induced by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Administration of CCK analogue (50 nmol/kg ip.) for 14 days treatment improved the locomotor and exploratory activity of mice, and improved bradykinesia and movement balance of mice. The CCK analogue administration also restored tyrosine hydroxylase (TH) positive dopaminergic neurons number and synapse number (synaptophysin levels) in the substantia nigra pars compacta (SNpc). The CCK analogue decreased glia activation and neuroinflammation in the SNpc, and regulated autophagy dysfunction induced by MPTP. CCK analogue protected against mitochondrial damage and ER stress, and also decreased the ratio of apoptosis signaling molecules Bax/Bcl-2. Importantly, the CCK analogue improved the decrease of p-CREBS133 growth factor signaling in the SNpc. Therefore, the CCK analogue promotes cell survival of dopaminergic neuron in the SNpc by activating the cAMP/PKA/CREB pathway that also inhibits apoptosis and regulates autophagy impairment. The present results indicate that CCK analogue shows a promising potential for the treatment of PD.

Induction of pancreatitis in mice with susceptibility to pancreatic cancer.

Chronic inflammation is known to be associated with pancreatic cancer, however a complete picture regarding how these pathologies intersect is still being characterized. In vivo model systems are critical for the study of mechanisms underlying how inflammation accelerates neoplasia. Repeat injection of cerulein, a cholecystokinin (CCK) analog, is widely used to experimentally induce acute and chronic pancreatitis in vivo. Chronic cerulein administration into genetically engineered mouse models (GEMMs) with predisposition to pancreatic cancer can induce a pro-inflammatory immune response, pancreatic acinar cell damage, pancreatic stellate cell activation, and accelerate the onset of neoplasia. Here we provide a detailed protocol and insights into using cerulein to induce pancreatitis in GEMMs, and methods to experimentally assess inflammation and pancreatic neoplasia.

TRPM4 links calcium signaling to membrane potential in pancreatic acinar cells

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is a Ca2+-activated nonselective cation channel that mediates membrane depolarization. Although, a current with the hallmarks of a TRPM4-mediated current has been previously reported in pancreatic acinar cells (PACs), the role of TRPM4 in the regulation of acinar cell function has not yet been explored. In the present study, we identify this TRPM4 current and describe its role in context of Ca2+ signaling of PACs using pharmacological tools and TRPM4-deficient mice. We found a significant Ca2+-activated cation current in PACs that was sensitive to the TRPM4 inhibitors 9-phenanthrol and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). We demonstrated that the CBA-sensitive current was responsible for a Ca2+-dependent depolarization of PACs from a resting membrane potential of −44.4 ± 2.9 to −27.7 ± 3 mV. Furthermore, we showed that Ca2+ influx was higher in the TRPM4 KO- and CBA-treated PACs than in control cells. As hormone-induced repetitive Ca2+ transients partially rely on Ca2+ influx in PACs, the role of TRPM4 was also assessed on Ca2+ oscillations elicited by physiologically relevant concentrations of the cholecystokinin analog cerulein. These data show that the amplitude of Ca2+ signals was significantly higher in TRPM4 KO than in control PACs. Our results suggest that PACs are depolarized by TRPM4 currents to an extent that results in a significant reduction of the inward driving force for Ca2+. In conclusion, TRPM4 links intracellular Ca2+ signaling to membrane potential as a negative feedback regulator of Ca2+ entry in PACs.

Astaxanthin Inhibits Interleukin-6 Expression in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells.

Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.

α-Lipoic Acid Inhibits IL-6 Expression by Upregulating PPAR-γ-Mediated Expression of Antioxidants in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells

ObjectivesOxidative stress is regarded as a major pathogenic factor in acute pancreatitis. Obesity is thought to be a negative prognostic factor in acute pancreatitis. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Recent study showed that resistin aggravates the expression of inflammatory cytokines such as interleukine-6 (IL-6) and production of reactive oxygen species (ROS) in pancreatic acinar cells stimulated with cerulein, a cholecystokinin analogue, as an in vitro acute pancreatitis model. Peroxisome proliferator-activated receptor (PPAR)-γ increases expression of antioxidant enzymes such as catalase and heme oxygenase-1 (HO-1). α-Lipoic acid is a powerful antioxidant and anti-inflammatory nutrient. The present study was purposed to investigate whether α-lipoic acid inhibits IL-6 expression in resistin/cerulein-stimulated pancreatic acinar cells and to determine whether it reduces ROS in AR42J cells by upregulating PPAR-γ-mediated expression of HO-1 and catalase in pancreatic acinar cells. MethodsRat pancreatic acinar cell line, AR42J cells, were stimulated with resistin (2 ng/ml) and cerulean (10–8 M), in the presence or absence of α-lipoic acid. mRNA expression of IL-6 was determined by real-time PCR analysis. ROS levels were measured using DCF-DA fluorescence. Expression of PPAR-γ, HO-1, and catalase were determined by Western blotting. Resultsα-Lipoic acid significantly decreased IL-6 mRNA expression and ROS production in resistin/cerulein-stimulated AR42J cells. α-Lipoic acid also increased expression of PPAR-γ, HO-1 and catalase. Inhibitory effect of α-lipoic acid on resistin/cerulein–induced IL-6 expression was suppressed by addition of a specific PPAR-γ inhibitor GW9662. GW9662 reversed the effect of α-lipoic acid on expression of HO-1 and catalase in AR42J cells. Conclusionsα-Lipoic acid suppresses cerulein/resistin-induced IL-6 expression and ROS production through PPAR-γ-mediated expression of HO-1 and catalase in pancreatic acinar cells. Funding SourcesThis study was supported by a Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

Low Incidence of High-Grade Pancreatic Intraepithelial Neoplasia Lesions in a Crmp4 Gene–Deficient Mouse Model of Pancreatic Cancer

Pancreatic intraepithelial neoplasia (PanIN), the most common premalignant lesion of the pancreas, is a histologically well-defined precursor to invasive pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms underlying the progression of PanINs have not been fully elucidated. Previously, we demonstrated that the expression of collapsin response mediator protein 4 (CRMP4) in PDAC was associated with poor prognosis. The expression of CRMP4 was also augmented in a pancreatitis mouse model. However, the role of CRMP4 in the progression of PanIN lesions remains uncertain. In the present study, we examined the relationship between CRMP4 expression and progression of PanIN lesions using genetically engineered mouse models. PanIN lesions were induced by peritoneal injection of the cholecystokinin analog caerulein in LSL-KRASG12D; Pdx1-Cre (KC-Crmp4 wild-type, WT) mice and LSL-KRASG12D; Pdx1-Cre; Crmp4−/− (KC-Crmp4 knockout, KO) mice. We analyzed pancreatic tissue sections from these mice and evaluated PanIN grade by hematoxylin and eosin staining. CRMP4 expression was examined and the cellular components assessed by immunohistochemistry using antibodies against CRMP4, CD3, and α-smooth muscle actin (SMA). The incidence of high-grade PanIN in KC-Crmp4 WT mice was higher than that in KC-Crmp4 KO animals. CRMP4 was expressed not only in epithelial cells but also in αSMA-positive cells in stromal areas of PanIN lesions. The CRMP4 expression in stromal areas correlated with PanIN grade in WT mice. These results suggested that the expression of CRMP4 in stromal cells may underlie the incidence or progression of PanIN.

Loss of RE-1 silencing transcription factor accelerates exocrine damage from pancreatic injury

Regulation of pancreas plasticity is critical for preventing injury and promoting regeneration upon tissue damage. The intricate process of pancreatic differentiation is governed by an orchestrated network of positive and negative transcription factors for appropriate gene expression. While the transcriptional repressor REST is well characterized as a silencer of neuronal genes in non-neuronal cells, the role of REST in regulating exocrine pancreas cell identity remains largely unexplored. Rest expression is increased upon injury in the mouse pancreas, such as induced acute and chronic pancreatitis and ductal adenocarcinoma. At the cellular level, Rest expression is lower in mature acinar cells compared with pancreas progenitor and ductal cells. To investigate the role of REST activity in pancreatic transdifferentiation and homeostasis, we developed a novel mouse model (Cre/RESTfl/fl) with conditional knockout (KO) of Rest expression within pancreas cells. The high Cre-mediated excision efficiency of Rest exon two KO caused decreased Rest expression and activity within the pancreas. Short-term organoid cultures of pancreatic acini to undergo acinar-to-ductal metaplasia (ADM) showed that loss of REST impedes induced ADM, while overexpression of REST increases ADM. Interestingly, REST ablation accelerated acute pancreatitis in mice treated with the cholecystokinin analog caerulein, as indicated by cellular morphology, elevated serum amylase levels and pancreatic edema. Furthermore, Cre/RESTfl/fl mice were more sensitive to acute pancreatitis injury and displayed augmented tissue damage and cellular lesions. These results suggest REST has a novel protective role against pancreatic tissue damage by acting as a regulator of exocrine cell identity.

Preventive Effects of Gardenia jasminoides on Cerulein-Induced Chronic Pancreatitis.

Our previous report revealed that Gardenia jasminoides (GJ) has protective effects against acute pancreatitis. So, we examined whether aqueous extract of GJ has anti-inflammation and antifibrotic effects even against cerulein-induced chronic pancreatitis (CP). CP was induced in mice by an intraperitoneal injection of a stable cholecystokinin (CCK) analogue, cerulein, six times a day, four days per week for three weeks. GJ extract (0.1 or 1[Formula: see text]g/kg) or saline (control group) were intraperitoneally injected 1[Formula: see text]h before first cerulein injection. After three weeks of stimulation, the pancreas was harvested for the examination of several fibrotic parameters. In addition, pancreatic stellate cells (PSCs) were isolated using gradient methods to examine the antifibrogenic effects of GJ. In the cerulein-induced CP mice, the histological features of the pancreas showed severe tissue damage such as enlarged interstitial spaces, inflammatory cell infiltrate and glandular atrophy, and tissue fibrosis. However, treatment of GJ reduced the severity of CP such as pancreatic edema and inflammatory cell infiltration. Furthermore, treatment of GJ increased pancreatic acinar cell survival, and reduced pancreatic fibrosis and activation of PSC in vivo and in vitro. In addition, GJ treatment inhibited the activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) in the PSCs. These results suggest that GJ attenuated the severity of CP and the pancreatic fibrosis by inhibiting JNK and ERK activation during CP.

Abstract 5222: Repression of neuronal genes protects the pancreas from certain injuries and aberrant plasticity

Abstract Among the major cancer types, pancreatic ductal adenocarcinoma has one of the lowest survival rates with little improvement over the past 3 decades. To address this dire clinical need, this study explores the unique and underexploited neuronal property of the pancreas that correlates with greater injury and pancreatic cancer development. The pancreas has a remarkable innate plasticity, and a shift toward neuroendocrine transdifferentiation has been shown to correlate with poor survival and tumor progression in numerous cancers, such as lung and prostate cancers. Recent reports show a subpopulation of cells within pancreatic tumors that express neuroendocrine-related genes correlate with poor outcome and chemoresistance. We hypothesize one regulator of this ductal-neuroendocrine lineage plasticity may be REST, a transcriptional repressor most known for orchestrating neuronal development. Tissue microarray of human pancreatic cancer shows patients negative for REST had a lower overall survival than patients positive for REST. To investigate the role of REST in the pancreas, we developed a novel transgenic mouse model with REST conditionally knocked out of the pancreas. REST KO resulted in an increase of neuroendocrine markers and an increase of zymogen granules, resulting in a lighter, cloudy appearance of the pancreas. When pancreatic injury was introduced by injections of cholecystokinin analog caerulein to induce acute pancreatitis, the REST KO mice show greater serum amylase levels and higher pancreatic edema indicating greater pancreatitis. Furthermore, an increase in neuronal-related genes was observed within REST KO pancreas relative to control. We then investigated the role of REST during cancer-promoting injuries such as chronic pancreatitis. Mice were administered a 10-week series of caerulein injections, and histology was evaluated. As expected, REST KO mice experienced greater inflammation, injury, and pre-neoplastic lesions. Further directions include investigating REST KO during later stages of pancreatic cancer injury by crossing REST KO mice with mutant KRAS mice, as oncogenic KRAS is a leading driver of pancreatic cancer. We expect REST KO to increase neuroendocrine properties that will accelerate KRAS-induced pancreatic cancer development. In conclusion, we hypothesize that loss of REST increases neuroendocrine-like properties of the pancreas that leads to a greater injury, suggesting that REST plays a protective effect against pancreatic damage and pancreatic cancer development. Furthermore, our novel REST KO mice may prove to be a useful model to investigate how neuroendocrine properties may promote pancreatic cancer development and drug resistance. Understanding neuronal influences during pancreatic cancer development could have multiple implications for cancer patients and warrants further investigation. Citation Format: Julie K. Bray, Ola Elgamal, Lais Da Silva, Dhruvit Sutaria, Xiuli Liu, Kristianna Fredenburg, Thomas D. Schmittgen. Repression of neuronal genes protects the pancreas from certain injuries and aberrant plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5222.

Effect of Docosahexaenoic Acid on Ca2+ Signaling Pathways in Cerulein-Treated Pancreatic Acinar Cells, Determined by RNA-Sequencing Analysis.

Intracellular Ca2+ homeostasis is commonly disrupted in acute pancreatitis. Sustained Ca2+ release from internal stores in pancreatic acinar cells (PACs), mediated by inositol triphosphate receptor (IP3R) and the ryanodine receptor (RyR), plays a key role in the initiation and propagation of acute pancreatitis. Pancreatitis induced by cerulein, an analogue of cholecystokinin, causes premature activation of digestive enzymes and enhanced accumulation of cytokines and Ca2+ in the pancreas and, as such, it is a good model of acute pancreatitis. High concentrations of the omega-3 fatty acid docosahexaenoic acid (DHA) inhibit inflammatory signaling pathways and cytokine expression in PACs treated with cerulein. In the present study, we determined the effect of DHA on key regulators of Ca2+ signaling in cerulein-treated pancreatic acinar AR42 J cells. The results of RNA-Sequencing (RNA-Seq) analysis showed that cerulein up-regulates the expression of IP3R1 and RyR2 genes, and that pretreatment with DHA blocks these effects. The results of real-time PCR confirmed that DHA inhibits cerulein-induced IP3R1 and RyR2 gene expression, and demonstrated that DHA pre-treatment decreases the expression of the Relb gene, which encodes a component of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional activator complex, and the c-fos gene, which encodes a component of activator protein-1 (AP-1) transcriptional activator complex. Taken together, DHA inhibits mRNA expression of IP3R1, RyR2, Relb, and c-fos, which is related to Ca2+ network in cerulein-stimulated PACs.

Efficient Synthesis of a Family of Bifunctional Chelators Based on the PCTA[12] Macrocycle Suitable for Bioconjugation

PCTA[12] is a 12‐membered tetraaza‐macrocyclic ligand that incorporates a pyridine unit within the macrocyclic ring and three acetate pendant arms. Unlike DOTA and NOTA chelators, PCTA is a recent entry to the field of macrocyclic polyaminocarboxylate ligands available to complex a variety of M2+/M3+ ions for biomedical applications such as diagnostic and radiotherapeutic. Despite the promising properties of its chelates, only a few of bifunctional chelating agents (BFCAs) derived from PCTA have been described so far. Based on our very recent methodology for the preparation of PCTA[12] itself, we report here the efficient synthesis of several BFCAs derived from PCTA bearing a free reactive function group, mainly devoted to conjugation purposes: ester, carboxylic acid, alcohol, aliphatic amine, aromatic amine, maleimide, bromo or azide functions. These functions were introduced either on the 4‐position of the pyridine ring or on the methylene carbon atom of the central acetate chelating arm, while keeping the three carboxylate groups available for metal chelation. Moreover, two of these BFCAs‐PCTA were used for conjugation with a tetrapeptide (cholecystokinin analogue), a bioactive molecule (biotin), or a solid support (silica gel).