Implicating the cholecystokinin B receptor in liver stem cell oncogenesis.

Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related deaths worldwide. Chronic inflammation and fibrosis are the greatest risk factors for the development of HCC. Although the cell of origin for HCC is uncertain, many theories believe this cancer may arise from liver progenitor cells or stem cells. Here, we describe the activation of hepatic stem cells that overexpress the cholecystokinin-B receptor (CCK-BR) after liver injury with either a DDC diet (0.1% 3, 5-diethoxy-carbonyl 1,4-dihydrocollidine) or a NASH-inducing CDE diet (choline-deficient ethionine) in murine models. Pharmacologic blockade of the CCK-BR with a receptor antagonist proglumide or knockout of the CCK-BR in genetically engineered mice during the injury diet reduces the expression of hepatic stem cells and prevents the formation of three-dimensional tumorspheres in culture. RNA sequencing of livers from DDC-fed mice treated with proglumide or DDC-fed CCK-BR knockout mice showed downregulation of differentially expressed genes involved in cell proliferation and oncogenesis and upregulation of tumor suppressor genes compared with controls. Inhibition of the CCK-BR decreases hepatic transaminases, fibrosis, cytokine expression, and alters the hepatic immune cell signature rendering the liver microenvironment less oncogenic. Furthermore, proglumide hastened recovery after liver injury by reversing fibrosis and improving markers of synthetic function. Proglumide is an older drug that is orally bioavailable and being repurposed for liver conditions. These findings support a promising therapeutic intervention applicable to patients to prevent the development of HCC and decrease hepatic fibrosis.NEW & NOTEWORTHY This investigation identified a novel pathway involving the activation of hepatic stem cells and liver oncogenesis. Receptor blockade or genetic disruption of the cholecystokinin-B receptor (CCK-BR) signaling pathway decreased the activation and proliferation of hepatic stem cells after liver injury without eliminating the regenerative capacity of healthy hepatocytes.

Treatment with a Cholecystokinin Receptor Antagonist, Proglumide, Improves Efficacy of Immune Checkpoint Antibodies in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated deaths worldwide. Treatment with immune checkpoint antibodies has shown promise in advanced HCC, but the response is only 15-20%. We discovered a potential target for the treatment of HCC, the cholecystokinin-B receptor (CCK-BR). This receptor is overexpressed in murine and human HCC and not in normal liver tissue. Mice bearing syngeneic RIL-175 HCC tumors were treated with phosphate buffer saline (PBS; control), proglumide (a CCK-receptor antagonist), an antibody to programmed cell death protein 1 (PD-1Ab), or the combination of proglumide and the PD-1Ab. In vitro, RNA was extracted from untreated or proglumide-treated murine Dt81Hepa1-6 HCC cells and analyzed for expression of fibrosis-associated genes. RNA was also extracted from human HepG2 HCC cells or HepG2 cells treated with proglumide and subjected to RNA sequencing. Results showed that proglumide decreased fibrosis in the tumor microenvironment and increased the number of intratumoral CD8+ T cells in RIL-175 tumors. When proglumide was given in combination with the PD-1Ab, there was a further significant increase in intratumoral CD8+ T cells, improved survival, and alterations in genes regulating tumoral fibrosis and epithelial-to-mesenchymal transition. RNAseq results from human HepG2 HCC cells treated with proglumide showed significant changes in differentially expressed genes involved in tumorigenesis, fibrosis, and the tumor microenvironment. The use of the CCK receptor antagonist may improve efficacy of immune checkpoint antibodies and survival in those with advanced HCC.

Proglumide Reverses Nonalcoholic Steatohepatitis by Interaction with the Farnesoid X Receptor and Altering the Microbiome.

Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in the livers of mice with NASH. The farnesoid X receptor (FXR) is involved in bile acid transport and is a target for novel therapeutics for NASH. The aim of this study was to examine the role of proglumide, a CCK receptor inhibitor, in a murine model of NASH and its interaction at FXR. Mice were fed a choline deficient ethionine (CDE) diet to induce NASH. Some CDE-fed mice received proglumide-treated drinking water. Blood was collected and liver tissues were examined histologically. Proglumide's interaction at FXR was evaluated by computer modeling, a luciferase reporter assay, and tissue FXR expression. Stool microbiome was analyzed by RNA-Sequencing. CDE-fed mice developed NASH and the effect was prevented by proglumide. Computer modeling demonstrated specific binding of proglumide to FXR. Proglumide binding in the reporter assay was consistent with a partial agonist at the FXR with a mean binding affinity of 215 nM. FXR expression was significantly decreased in livers of CDE-fed mice compared to control livers, and proglumide restored FXR expression to normal levels. Proglumide therapy altered the microbiome signature by increasing beneficial and decreasing harmful bacteria. These data highlight the potential novel mechanisms by which proglumide therapy may improve NASH through interaction with the FXR and consequent alteration of the gut microbiome.

Cholecystokinin Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis.

BACKGROUND AND AIMS: Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. METHODS: Two murine models were used to determine whether CCK receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18 weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. RESULTS: Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFßR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. CONCLUSION: New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.

A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. METHODS: We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient-ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. RESULTS: CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. CONCLUSION: These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.

UCN3 suppresses food intake in coordination with CCK and the CCK2R in Siberian sturgeon (Acipenser baerii).

Urocortin-3 (UCN3) as a brain-gut peptide inhibits food intake of animal, but the underlying mechanism is not clear. To explore the appetite mechanism about the action of UCN3 in fish, intraperitoneal injection of UCN3 with CCK8, Lorglumide (CCK1R antagonist) or LY225910 (CCK2R antagonist) were conducted. Siberian sturgeon administrated with UCN3 and CCK8 showed a drastic reduction in food intake. The anorectic effect of UCN3 was significantly blocked by LY225910, but not affected by Lorglumide. Furthermore, LY225910 could effectively reverse appetite factor mRNA expressions, including cck, pyy, cart, npy, ucn3, apelin and nucb2 in the whole brain, stomach and intestinum valvula, but Lorglumide could only partially reverse these effects, suggesting the anorectic effect of UCN3 may be primarily mediated CCK2R in Siberian sturgeon. This study indicates for the first time in fish that UCN3 may inhibit food intake in coordination with CCK and CCK2R.

Dietary fat stimulates pancreatic cancer growth and promotes fibrosis of the tumor microenvironment through the cholecystokinin receptor.

The gastrointestinal peptide cholecystokinin (CCK) is released from the duodenum in response to dietary fat to aid in digestion, and plasma CCK levels are elevated with the consumption of high-fat diets. CCK is also a trophic peptide for the pancreas and has also been shown to stimulate growth of pancreatic cancer. In the current investigation, we studied the influence of a diet high in saturated fat on the growth of pancreatic cancer in syngeneic murine models before the mice became obese to exclude the confounding factors associated with obesity. The high-fat diet significantly increased growth and metastasis of pancreatic cancer compared with the control diet, and the stimulatory effect was blocked by the CCK-receptor antagonist proglumide. We then selectively knocked out the CCK receptor on the pancreatic cancer cells using clustered regularly interspaced short palindromic repeats technology and showed that without CCK-receptors, dietary fat was unable to stimulate cancer growth. We next demonstrated that dietary fat failed to influence pancreatic cancer xenograft growth in genetically engineered CCK peptide knockout mice. The tumor-associated fibrosis that is so prevalent in the pancreatic cancer microenvironment was significantly decreased with CCK-receptor antagonist therapy because fibroblasts also have CCK receptors. The CCK-receptor antagonist proglumide also altered tumor metalloprotease expression and increased tumor suppressor genes by a PCR array. Our studies confirm that a diet high in saturated fat promotes growth of pancreatic cancer and the action is mediated by the CCK-receptor pathway. NEW & NOTEWORTHY Diets high in long-chain saturated fats promote growth of pancreatic cancer independent of obesity. The mechanism through which dietary fat promotes cancer is mediated through the cholecystokinin (CCK) receptor pathway. Therapy with a CCK-receptor antagonist altered the tumor microenvironment by reducing fibrosis, increasing cluster of differentiation 8+ lymphocytes, increasing tumor suppressor genes, and thus decreasing metastases. Use of CCK-receptor antagonist therapy with standard chemotherapy for pancreatic cancer may improve response by altering the tumor microenvironment.

Activation of galanin and cholecystokinin receptors in the lumbosacral spinal cord is required for ejaculation in male rats.

The spinal ejaculation generator is comprised of lumbar spinothalamic (LSt) cells and their axonal projections to autonomic and motor neurons in the lumbosacral spinal cord. LSt cells regulate ejaculatory reflexes by release of neuropeptides that are co-expressed in their axons, as previously demonstrated for gastrin-releasing peptide and enkephalin. Here, the role of two other neuropeptides co-expressed in LSt cells for ejaculatory reflexes is demonstrated: galanin and cholecystokinin (CCK). Adult male rats were anesthetized, spinalized, and received intrathecal infusions of galanin receptor antagonist Galantide (1 or 10 nmol) or CCK receptor antagonist proglumide (71 or 714 nmol). The dorsal penile nerve (DPN) was electrically stimulated to trigger ejaculatory reflexes and seminal vesicle pressure (SVP) and rhythmic contractions of the bulbocavernosus muscle (BCM) were analyzed as parameters of emission and expulsion respectively. Treatment with galanin or CCK antagonists significantly reduced SVP increases and BCM bursting, demonstrating that galanin and CCK are required for ejaculation. Next, anesthetized, spinalized males received intrathecal infusions of galanin (0.15 or 0.3 nmol) or CCK(26-33) (4.35 nmol) and effects on subthreshold DPN stimulations were determined. Intrathecal infusions of galanin or CCK facilitated ejaculatory reflexes induced by subthreshold DPN stimulation in all animals, but did not trigger ejaculatory reflexes in the absence of DPN stimulation. Together, these results demonstrate that galanin and CCK both act in the spinal ejaculation generator to regulate ejaculation. However, effects of galanin and CCK were dependent on DPN stimulation, suggesting that these neuropeptides may act in concert with other LSt co-expressed neuropeptides.

Lack of Analgesic Synergy of the Cholecystokinin Receptor Antagonist Proglumide and Spinal Cord Stimulation for the Treatment of Neuropathic Pain in Rats.

OBJECTIVE: Neuropathic pain is difficult to manage and treat. Spinal cord stimulation (SCS) has become an established procedure for treating chronic neuropathic pain that is refractory to pharmacological therapy. In order to achieve better analgesia, a number of studies have evaluated the effectiveness of combining drug therapy with SCS. Cholecystokinin antagonists, such as proglumide, enhance the analgesic efficacy of endogenous opioids in animal models of pain. We previously reported that both systemic and spinal administration of proglumide enhances analgesia produced by both low- and high-frequency transcutaneous electrical nerve stimulation (TENS). Since SCS produces analgesia through endogenous opioids, we hypothesized that the analgesic effect of SCS would be enhanced through co-administration with proglumide in animals with neuropathic pain. MATERIALS AND METHODS: Male Sprague-Dawley rats (n = 40) with spared nerve injury were given proglumide (20 mg/kg, i.p.) or saline prior to treatment with SCS (sham, 4 Hz, and 60 Hz). Mechanical withdrawal thresholds of the paw were measured before and after induction of nerve injury, and after SCS. Physical activity levels were measured after SCS. RESULTS: Both proglumide and SCS when given independently significantly increased withdrawal thresholds two weeks after nerve injury. However, there was no additional effect of combining proglumide and SCS on mechanical withdrawal thresholds or activity levels in animals with nerve injury. DISCUSSION AND CONCLUSIONS: Proglumide may be a candidate for achieving analgesia for patients with refractory neuropathic pain conditions, but does not enhance analgesia produced by SCS.

Postsynaptic Depolarization Enhances GABA Drive to Dorsomedial Hypothalamic Neurons through Somatodendritic Cholecystokinin Release.

Somatodendritically released peptides alter synaptic function through a variety of mechanisms, including autocrine actions that liberate retrograde transmitters. Cholecystokinin (CCK) is a neuropeptide expressed in neurons in the dorsomedial hypothalamic nucleus (DMH), a region implicated in satiety and stress. There are clear demonstrations that exogenous CCK modulates food intake and neuropeptide expression in the DMH, but there is no information on how endogenous CCK alters synaptic properties. Here, we provide the first report of somatodendritic release of CCK in the brain in male Sprague Dawley rats. CCK is released from DMH neurons in response to repeated postsynaptic depolarizations, and acts in an autocrine fashion on CCK2 receptors to enhance postsynaptic NMDA receptor function and liberate the retrograde transmitter, nitric oxide (NO). NO subsequently acts presynaptically to enhance GABA release through a soluble guanylate cyclase-mediated pathway. These data provide the first demonstration of synaptic actions of somatodendritically released CCK in the hypothalamus and reveal a new form of retrograde plasticity, depolarization-induced potentiation of inhibition. Significance statement: Somatodendritic signaling using endocannabinoids or nitric oxide to alter the efficacy of afferent transmission is well established. Despite early convincing evidence for somatodendritic release of neurohypophysial peptides in the hypothalamus, there is only limited evidence for this mode of release for other peptides. Here, we provide the first evidence for somatodendritic release of the satiety peptide cholecystokinin (CCK) in the brain. We also reveal a new form of synaptic plasticity in which postsynaptic depolarization results in enhancement of inhibition through the somatodendritic release of CCK.

Transcriptomic and behavioural characterisation of a mouse model of burn pain identify the cholecystokinin 2 receptor as an analgesic target.

Burn injury is a cause of significant mortality and morbidity worldwide and is frequently associated with severe and long-lasting pain that remains difficult to manage throughout recovery. We characterised a mouse model of burn-induced pain using pharmacological and transcriptomic approaches. Mechanical allodynia elicited by burn injury was partially reversed by meloxicam (5 mg/kg), gabapentin (100 mg/kg) and oxycodone (3 and 10 mg/kg), while thermal allodynia and gait abnormalities were only significantly improved by amitriptyline (3 mg/kg) and oxycodone (10 mg/kg). The need for relatively high opioid doses to elicit analgesia suggested a degree of opioid resistance, similar to that shown clinically in burn patients. We thus assessed the gene expression changes in dorsal root ganglion neurons and pathophysiological mechanisms underpinning burn injury-induced pain using a transcriptomic approach. Burn injury was associated with significantly increased expression of genes associated with axon guidance, neuropeptide signalling, behavioural defence response and extracellular signalling, confirming a mixed neuropathic and inflammatory aetiology. Notably, among the pain-related genes that were upregulated post-injury was the cholecystokinin 2 receptor (Cckbr), a G protein-coupled receptor known as a pain target involved in reducing opioid effectiveness. Indeed, the clinically used cholecystokinin receptor antagonist proglumide (30 mg/kg) was effective at reversing mechanical allodynia, with additional analgesia evident in combination with low-dose oxycodone (1 mg/kg), including significant reversal of thermal allodynia. These findings highlight the complex pathophysiological mechanisms underpinning burn injury-induced pain and suggest that cholecystokinin-2 receptor antagonists may be useful clinically as adjuvants to decrease opioid requirements and improve analgesic management.

Ameliorating effects of proglumide on neurobehavioral and biochemical deficits in animal model of status epilepticus.

Status epilepticus (SE) is a recurrent generalized convulsion condition and is regarded as a medical emergency with around 50% of the cases occurring in children. Besides neurobehavioral and motor deficits, SE is reportedly associated with imbalance in a number of neurochemicals in several areas of the brain. Furthermore, neuronal hyperactivity and/or excitotoxicity in such brain areas have been associated with excessive generation of free radicals. Proglumide (Pgm) is a known cholecystokinin (CCK) antagonist and any changes in the level of CCK and in the number of CCK receptors has been linked with SE. The present study was designed to investigate the possible neuroprotective effects of Pgm (0, 250, 500 and 750mg/ml/kg i.p.) on epileptic seizure activities, some neurobehavioral tests, and on some oxidative stress related parameters like lipid peroxides measured as thiobarbituric acid-reactive substance (TBARS) and total glutathione (GSH) in brain (hippocampus and striatum) of young rats that were experimentally induced with SE by lithium (Li) in 3mEq/ml/kg dose, i.p. followed 20h later by pilocarpine (Pc) in 20mg/ml/kg dose, s.c.). Besides significant anti-epileptic effect, Pgm significantly ameliorated SE-induced deterioration in cognitive behavior (in water-maze), motor performance (on rotarod), and biochemical changes in brain. It is concluded from the present study that Pgm has significant neuroprotective effects against SE and this effect may probably be due to its antioxidant activity. Pgm may prove to be a potentially effective antiepileptic drug, however, further studies are needed to ascertain this possibility.

The long chain fatty acid oleate activates mouse intestinal afferent nerves in vitro.

Vagal afferents innervating the gastrointestinal tract serve an important nutrient-sensing function, and these signals contribute to satiety. Detection of nutrients occurs largely through the release of mediators from specialized enteroendocrine cells within the mucosa of the gastrointestinal tract. The signaling pathways leading to vagal afferent activation are not clear; however, previous in-vivo studies have implicated a role for cholecystokinin (CCK). We used an in vitro intestinal afferent extracellular recording preparation to study the effect of luminal perfusion of the long chain fatty acid oleate on mouse intestinal afferent activity. Oleate activated intestinal afferents in a concentration-dependent fashion, with an EC50 value of approximately 25 mmol/L. The L-type calcium channel blocker nicardipine attenuated the effect of oleate. Vagotomy resulted in a significant (>60%) reduction of the responses to both oleate and CCK. The CCK-1 receptor antagonist lorglumide nearly abolished responses to CCK and oleate. Our experiments therefore suggest that oleate activates intestinal afferents, with vagal afferents primarily involved; however, nonvagal fibres also contribute. The activation is dependent on CCK release, likely via activation of L-type channels on mucosal enteroendocrine cells, finally resulting in activation of CCK-1 receptors on the afferent terminals.

Humoral regulation of heart rate during digestion in pythons (Python molurus and Python regius).

Pythons exhibit a doubling of heart rate when metabolism increases several times during digestion. Pythons, therefore, represent a promising model organism to study autonomic cardiovascular regulation during the postprandial state, and previous studies show that the postprandial tachycardia is governed by a release of vagal tone as well as a pronounced stimulation from nonadrenergic, noncholinergic (NANC) factors. Here we show that infusion of plasma from digesting donor pythons elicit a marked tachycardia in fasting snakes, demonstrating that the NANC factor resides in the blood. Injections of the gastrin and cholecystokinin receptor antagonist proglumide had no effect on double-blocked heart rate or blood pressure. Histamine has been recognized as a NANC factor in the early postprandial period in pythons, but the mechanism of its release has not been identified. Mast cells represent the largest repository of histamine in vertebrates, and it has been speculated that mast cells release histamine during digestion. Treatment with the mast cell stabilizer cromolyn significantly reduced postprandial heart rate in pythons compared with an untreated group but did not affect double-blocked heart rate. While this study indicates that histamine induces postprandial tachycardia in pythons, its release during digestion is not stimulated by gastrin or cholecystokinin nor is its release from mast cells a stimulant of postprandial tachycardia.

Morphological evidence that pentagastrin regulates secretion in the human parotid gland.

Salivary secretion is principally regulated by autonomic nerves. However, recent evidence from in vivo animal experiments suggests that gastrointestinal peptide hormones can also influence saliva production. The aim of the present study was to define the secretagogue activity of the gastrin-analogue pentagastrin in human salivary glands. For this purpose, parotid tissues were exposed to pentagastrin in vitro. Morphological techniques were used to evaluate modifications to serous acinar cells associated with secretion. Using a variant of the osmium maceration method, high resolution scanning electron microscopy allowed assessment of the morphology of the cytoplasmic aspect of the plasmalemma to demonstrate secretory activity. To quantify responses to pentagastrin, we recorded morphometric data on microvilli, microbuds, and protrusions. Dose-dependent morphological changes were observed, whereas protein concentration increased in the incubate. The use of selective receptor antagonists showed pentagastrin to act principally via cholecystokinin-A receptors. The morphological responses observed following exposure to pentagastrin differed from those elicited following exposure to the pan-muscarinic agonist carbachol. This study provides the first demonstration of a direct secretory action of gastrointestinal peptides on salivary glands in humans.

Safety and Dosing Study of a Cholecystokinin Receptor Antagonist in Non-alcoholic Steatohepatitis.

High saturated fat diets have been shown to raise blood levels of cholecystokinin (CCK) and induce nonalcoholic steatohepatitis (NASH). CCK receptors are expressed on stellate cells and are responsible for hepatic fibrosis when activated. The purpose of this study was to test the safety and dose of a CCK receptor antagonist, proglumide, in human participants with NASH. An open-label single ascending dose study was conducted in 18 participants with clinical NASH based upon steatosis by liver ultrasound, elevated hepatic transaminases, and a component of the metabolic syndrome. Three separate cohorts (N = 6 each) were treated with oral proglumide for 12 weeks in a sequential ascending fashion with 800 (Cohort 1), 1,200 (Cohort 2), and 1,600 (Cohort 3) mg/day, respectively. Blood hematology, chemistries, proglumide levels, a biomarker panel for fibrosis, and symptom surveys were determined at baseline and every 4 weeks. Abdominal ultrasounds and transient elastography utilizing FibroScan were obtained at baseline and at Week 12. Proglumide was well tolerated at all doses without any serious adverse events. There was no change in body weight from baseline to Week 12. For Cohorts 1, 2, and 3, the median percent change in alanine aminotransferase was 8.42, -5.05, and -22.23 and median percent change in fibrosis score by FibroScan was 8.13, -5.44, and -28.87 (kPa), respectively. Hepatic steatosis as measured by controlled attenuation parameter score significantly decreased with proglumide, (P < 0.05). Blood microRNA biomarkers and serum 4-hydroxyproline were consistent with decreased fibrosis at Week 12 compared with baseline. These findings suggest proglumide exhibits anti-inflammatory and anti-fibrotic properties and this compound is well tolerated in participants with NASH.

Targeting the Cholecystokinin Receptor: A Novel Approach for Treatment and Prevention of Hepatocellular Cancer.

Hepatocellular carcinoma (HCC) is the fastest growing cancer worldwide in part due to the obesity epidemic and fatty liver disease, particularly nonalcoholic steatohepatitis (NASH). Chronic inflammation with the release of cytokines and chemokines with activation of hepatic stellate cells results in changes of the liver extracellular matrix (ECM) that predisposes to the development of HCC. Blood levels of the gastrointestinal peptide cholecystokinin (CCK) are increased in humans and mice consuming a high-fat diet. We found that the CCK-B receptor (CCK-BR) expression increased in the livers of mice with NASH. Treatment of mice with a CCK-BR antagonist, proglumide, prevented NASH, lowered hepatic inflammatory cytokines and chemokines, reduced oxidative stress, decreased F4/80+ hepatic macrophages, and prevented HCC. CCK-AR and CCK-BR expression was increased in both murine and human HCC cell lines compared with that of normal liver, and CCK stimulated the growth of wild-type and CCK-A receptor knockout HCC cells in vitro, but not CCK-BR knockout cells suggesting that the CCK-BR mediates proliferation. Proglumide therapy significantly reduced growth by 70% and 73% in mice bearing Dt81Hepa1-6 or in RIL-75 HCC tumors, respectively. IHC of a human liver tissue array with a selective CCK-BR antibody revealed staining of human HCC and no staining in normal liver. PREVENTION RELEVANCE: This investigation demonstrates the role of the gastrointestinal peptide cholecystokinin (CCK) in hepatocellular carcinoma (HCC) and how CCK-BR blockade reverses the premalignant state of the hepatic extracellular matrix hence, rendering it less susceptible to the development of HCC. Thereby, CCK-BR blockade is a novel approach for the prevention/treatment of HCC.

CCK mediated the inhibitory effect of oxytocin on the contraction of longitudinal muscle strips of duodenum in male rats.

The aim of the present study was to investigate the effect of oxytocin (OT) on duodenum motility in rats and the possibility that cholecystokinin (CCK) was involved in this process. The isometric contraction of longitudinal muscle strips of duodenum was monitored by polygraph. ELISA was used to measure the concentration of CCK and OT in duodenum. CCK mRNA was assayed by RT-PCR. Oxytocin receptor (OTR) and CCK in duodenum were located by immunohistochemistry and immunofluorescence staining. OT (10⁻5 and 10⁻6 M) inhibited the spontaneous contraction of the muscle strips. On the contrary, atosiban (OT receptor antagonist), lorglumide (CCK1 receptor antagonist), and tetrodotoxin (TTX, blocker of voltage-dependent Na(+) channel on nerve fiber) excited the contraction. The inhibitory effect of OT on duodenal motility was reversed by pretreatment of atosiban, lorglumide, or TTX. Exogenous OT did not influence the expression of OT mRNA in duodenum but increased the concentration of CCK in the culture medium of the cells isolated from longitudinal muscle myenteric plexus. The OTR and CCK were co-expressed in the neurons of the myenteric plexus in duodenum. We concluded that OT inhibited the contraction of the LD spontaneous contraction of rats in vitro. This effect was mediated by the CCK released from the neurons of the myenteric plexus in duodenum.

Gastric leptin: a novel role in cardiovascular regulation.

Gastric-derived leptin affects satiety and gastrointestinal function via vagal mechanisms and has been shown to interact with the gut hormone cholecystokinin (CCK). CCK selectively inhibits splanchnic sympathetic nerve discharge (SND) and the activity of a subset of presympathetic vasomotor neurons in the rostroventrolateral medulla (RVLM). The present study sought to examine the effects of gastric leptin on arterial pressure (AP), heart rate (HR), SND, and RVLM neuronal activity to determine whether its effects on cardiovascular regulation are dependent on CCK(1) receptors and vagal afferent transmission. To mimic gastric leptin, leptin (15-30 microg/kg) was administered close to the coeliac artery in anesthetized, artificially ventilated Sprague-Dawley rats. Within 5 min, leptin selectively decreased the activity of RVLM neurons also inhibited by CCK (-27 +/- 4%; P < 0.001; n = 15); these inhibitory effects were abolished following administration of the CCK(1) receptor antagonist lorglumide. Leptin significantly decreased AP and HR (-10 +/- 2 mmHg, P < 0.001; and -8 +/- 2 beats/min, P < 0.01; n = 35) compared with saline (-1 +/- 2 mmHg, 3 +/- 2 beats/min; n = 30). In separate experiments, leptin inhibited splanchnic SND compared with saline (-9 +/- 2% vs. 2 +/- 3%, P < 0.01; n = 8). Bilateral cervical vagotomy abolished the sympathoinhibitory, hypotensive, and bradycardic effects of leptin (P < 0.05; n = 6). Our results suggest that gastric leptin may exert acute sympathoinhibitory and cardiovascular effects via vagal transmission and CCK(1) receptor activation and may play a separate role to adipose leptin in short-term cardiovascular regulation.

Stimulatory effect of N-methyltyramine, a congener of beer, on pancreatic secretion in conscious rats.

BACKGROUND: Alcoholic beverages stimulate gastric acid secretion and increase the appetite. Although ingested ethanol stimulates pancreatic secretion, alcoholic beverages contain several congeners. N-methyltyramine (NMT) was isolated from beer as a factor in stimulating gastric acid secretion. In this study, we examined NMT to determine whether the congener stimulated pancreatic secretion in conscious rats. METHODS: Cannulae were inserted into male Wistar rats to separately drain bile and pancreatic secretions: 2 duodenal cannulae, a gastric cannula, and an external jugular vein cannula. The rats were placed in modified Bollman-type restraint cages. After a 4-day recovery period, experiments were conducted on unanesthetized rats. Different concentrations of NMT (5, 25, and 50 microg/kg) solutions were infused into the stomach. To examine the mechanism, the effects of the proton pump inhibitor, cholecystokinin (CCK-BR) antagonist (YM022), CCK-AR antagonist (CR1505), and atropine were administered prior to the NMT (25 microg/kg) infusion. The effect of intravenous infusion of NMT (7.5 microg/kg) was then determined. Moreover, dispersed acini were prepared, and the effect of different concentrations of NMT on amylase release was determined. RESULTS: Intragastric administration of NMT significantly increased pancreatic exocrine secretion in a dose-dependent manner. Atropine eliminated the stimulatory effect of NMT, but the infusion of the proton pump inhibitor, YM022, and CR1505 did not. Intravenous infusion of NMT did not affect pancreatic secretion, and NMT did not stimulate amylase release in vitro. CONCLUSIONS: N-methyltyramine stimulates pancreatic secretion via the cholinergic gastro-pancreatic reflex. The NMT content in beer was 2 mg/l, so that if a person weighing 60 kg consumes a 750 ml of beer, 25 microg/kg NMT will be ingested. Therefore, the stimulatory effect of beer on pancreatic secretion was produced not only by ethanol but also by the congener, NMT.