Secretion In Mice Research Articles

A2B adenosine receptor inhibition by the dihydropyridine calcium channel blocker nifedipine involves colonic fluid secretion

The adenosine A2B receptor is a critical protein in intestinal water secretion. In the present study, we screened compound libraries to identify inhibitors of the A2B receptor and evaluated their effect on adenosine-induced intestinal fluid secretion. The screening identified the dihydropyridine calcium antagonists nifedipine and nisoldipine. Their respective affinities for the A2B receptor (Ki value) were 886 and 1,399 nM. Nifedipine and nisoldipine, but not amlodipine or nitrendipine, inhibited both calcium mobilization and adenosine-induced cAMP accumulation in cell lines. Moreover, adenosine injection into the lumen significantly increased fluid volume in the colonic loop of wild-type mice but not A2B receptor-deficient mice. PSB-1115, a selective A2B receptor antagonist, and nifedipine prevented elevated adenosine-stimulated fluid secretion in mice. Our results may provide useful insights into the structure–activity relationship of dihydropyridines for A2B receptor. As colonic fluid secretion by adenosine seems to rely predominantly on the A2B receptor, nifedipine could be a therapeutic candidate for diarrhoea-related diseases.

Abstract P5-04-22: Immune responses triggered by cryoablation of breast cancers

Abstract Background: Cryoablation, the destruction of cells by ultra-low temperatures, has been used to treat benign breast disease and two clinical trials (ACOSOG Z1072 and FROST) have been conducted to determine its utility in invasive breast cancers. The focus of the trials has been the rate of complete tumor ablation with no assessment of immunological responses. We hypothesize that neoantigens released during cryoablation might be sufficient to trigger a robust immune response to prevent and/or reduce spread and relapse of breast cancers. In this pilot study we 1) evaluated biopsy material, cryoablated specimens and axillary lymph nodes from patients with cancers smaller than 2cm enrolled in the ACOSOG Z1072 trial at Lankenau Medical Center [N=18] and 2) assessed immune responses and effects on metastases formation in the classical mouse mammary tumor model 4T1 in immune competent Balb/c mice. In both settings responses were compared to patients/mice treated with surgical resection alone. Methods: After obtaining IRB approval for retrospective analyses of specimens from the ACOSOG Z1072 trial, immunohistochemical staining of surgical specimens was performed. Sections were stained for CD4, CD8, CD20, CD21, and CD1c. In the IACUC approved animal experiments, 4T1 cells were injected orthotopically in the mammary fatpad to initiate tumor growth. Small tumors were treated by cryoablation or surgery alone. Animals were euthanized 7 days post-treatment and tissues were collected to assess cytokine levels and presence of dissociated 4T1 cells. Single-cell suspensions of tumor, tumor-draining lymph node [TDLN], and spleen were tested for secretion of mouse Th1/Th2 cytokines using a bead array and measured by flow cytometry. Possible metastatic spread was assessed by a clonogenic assay using cells from venous blood, lung and brain. Cell suspensions were seeded in growth medium with the selection agent 6-thioguanine, allowing only resistant 4T1 cells to form colonies. Results: Cryoablation transformed tumors in both patients and mice into a gelatinous mass surrounded by a fibrotic capsule. Sections of tumors from both humans and mice displayed a necrotic core and infiltrating lymphocytes in the microenvironment. The cryoablated human tumors had slightly higher presence of lymphocytes positive for CD8+ compared to CD4+. The inverse relation was observed in non-cryoblated specimens. No significant difference was observed for CD20+ lymphocytes. Tumor-draining lymph nodes from cryoablated patients had an elevated presence of CD20+ B cells compared to patients treated by surgery alone. Follicular dendritic cells (CD21+) were also present at higher numbers in TDLN from cryoablated patients. Animals treated with cryoablation displayed robust increases of Th1 and Th2 cytokines in both spleen and TDLN compared to animals with surgery treatment. In the animals, circulating tumor cells were found prior to treatment, while no 4T1 colonies formed from cell suspensions of lung and brain tissue [N=8]. At end-point, the surgery group had more 4T1 foci formed from lung and brain [mean foci /animal = 6.25 and 0.75, respectively; N=6] than the cryo group that had 2.25 and 0 foci in lung and brain, respectively [N=8]. Conclusion: Cryoablation of breast cancer lesions can induce stimulatory immune responses in vivo. These immune responses might explain why animals treated with cryoablation, though having circulating tumor cells at the time of treatment, exhibited fewer micro metastatic growths compared to surgery alone. The presence of elevated numbers of CD20+ in TDLN has been associated with improved disease-free survival. All local patients in the clinical trial are currently disease-free (5 to 9-year F/U) which is higher than expected recurrence rate at ~15% at 9 years post treatment. Citation Format: Allison A Campoverde, Jonah D Klein, Zachary Aukers, John Kennedy, Vlasta Zemba-Palko, Vincenzo Ciocca, Robin M Ciocca, Jennifer L Sabol, Ned Z Carp, Margaretha Wallon. Immune responses triggered by cryoablation of breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-22.

Acute glucose load induced islet β cells dysfunction in TLR4 dependent manner in male mice

Recent studies highlighted the significance of chronic inflammation, which is mediated in part by toll-like receptors 4 (TLR4), in islet β cell dysfunction by high-glucose exposure. However, about it is unclear whether islet β cell dysfunction in response to high glucose is associated with TLR4. This investigation was designed to address the effect of TLR4 deficiency on insulin secretion in mice in response to acute intravenous glucose load. Hyperglycemic clamp was used to impair insulin secretion, and intraperitoneal glucose tolerance test was carried out to analyze insulin secretion function of islet β cells. Our results showed that TLR4 deficiency repressed insulin secretion impairment in response to acute intravenous glucose load. Compared to wild-type mice, TLR4−/− mice did not exhibit increase of IL-1β and TNF-α level in plasma and pancreatic tissue in response to acute intravenous load of high glucose. However, recombinant IL-1β or TNF-α administration restored insulin secretion impairment induced by high glucose in TLR4−/− mice. Taken together, our results demonstrated that TLR4 activation and subsequent IL-1β and TNF-α production contribute to islet β cell dysfunction in mice in response to acute intravenous load of high glucose, which may provide a theoretical basis for diabetes complication improvement by physical exercise.

Effects of SFRP4 overexpression on the production of adipokines in transgenic mice

ABSTRACT Secreted frizzled-related protein (SFRP) 4 is an extracellular antagonist of Wnt signalling that regulates adipogenesis, and is highly in the visceral adipose tissue of obese individuals. However, it is still unclear how exactly SFRP4 regulates the secretion of adipokines in the adipose tissue in vivo, an event that is closely related to the pathogenesis of obesity and insulin resistance. In this study, we generated transgenic (Tg) mice overexpressing SFRP4 in the liver and investigated SFRP4 role in adipokine secretion in mice on a regular normal diet. In Tg mice, SFRP4 protein was overexpressed in the liver, as compared to wild-type littermates (non-Tg), and released into the blood. Moreover, the size of adipocytes was smaller in the visceral adipose tissue of Tg mice compared to controls. Additionally, SFRP4 overexpression affected the expression of genes related to adipocyte differentiation, causing the upregulation of adiponectin and glucose transporter 4, and the downregulation of CCAAT/enhancer-binding protein-β, in both visceral and subcutaneous adipose tissue. However, there was no difference in body weight or body composition between Tg and non-Tg mice. In summary, our data showed that SFRP4 overexpression altered adipocyte size and adipokine secretion, possibly affecting adipocyte differentiation, obesity, and glucose metabolism.

Hepatic Fat Content Is Associated with Fasting-Induced Fibroblast Growth Factor 21 Secretion in Mice Fed Soy Proteins.

Previous studies suggest that circulating fibroblast growth factor 21 (FGF21) levels are elevated in patients with fatty liver, while fasting-induced secretion of FGF21 is lower in obese patients. It has been reported that soy protein prevents hepatic fat accumulation and induces FGF21 secretion. The present study was designed to evaluate the response of circulating FGF21 levels to feeding and fasting in mice fed soy protein-rich diets. For this, C57BL/6J mice were distributed into control, high-fat high-sucrose (HFHS)-casein protein, HFHS-soy protein, and HFHS-β-conglycinin diet groups. Plasma samples were collected after 10 and 11 wk either in dark periods with feeding conditions or light periods under fasting conditions using a crossover design. After a 12-wk period of feeding, HFHS-induced hepatic fat accumulation was significantly reduced in the groups fed HFHS-soy protein and HFHS-β-conglycinin as compared to that in the HFHS-casein-fed group (p<0.05). Plasma FGF21 concentration was significantly higher in the dark/feeding periods in the HFHS-casein group (p<0.05), while in the HFHS-β-conglycinin group it was higher in the light/fasting periods (p<0.05). The amount of mesenteric fat was significantly lower in the HFHS-β-conglycinin group than in the HFHS-casein and HFHS-soy protein groups (p<0.01). The fasting-induced FGF21 secretion was significantly and negatively correlated with hepatic fat content (p<0.05). The present study revealed that hepatic fat accumulation was associated with lower fasting-induced FGF21 secretion, which was regulated better by dietary intake of soy protein. These results support the preventive effects of soy protein on central obesity.

Cortex Phellodendri extract’s anti-diarrhea effect in mice related to its modification of gut microbiota

Cortex Phellodendri extract (CPE) has been used in China to treat diarrhea whereas the underlying mechanisms remain poorly understood. Given that dysbiosis of gut microbiota is a potential reason for diarrhea, and that oral CPE has a low absorption rate in intestine, we hypothesized that modification of gut microbiota is an important factor in CPE’s anti-diarrhea effect. To test this hypothesis, we established a diarrhea model by challenging post-weaning mice with oral Enterotoxigenic-Escherichia coli (ETEC), and then the mice were treated with two doses of CPE (80 mg/kg bodyweight and 160 mg/kg bodyweight) or the vehicle control (phosphate buffered saline). Diarrhea indices, inflammatory factors, morphology of jejunum, short-chain fatty acids (SCFAs), and serum endocrine were determined. Modification of gut microbiota was analyzed using 16S rDNA high-throughput sequencing. The changes in functional profiles of gut microbiota were predicted using software PICRUSt. We then explored the association between CPE-responding bacteria and the symptoms indices with the spearman’s rank correlation coefficient and significance test. Compared with diarrheal mice, CPE decreased Gut/Carcass ratio and water content of stool, increased goblet cell density and villus height/crypt depth of jejunum, as well as decreased inflammatory indices (Tumour Necrosis Factor-α, Myeloperoxidase and Interleukin-1α). CPE shifted the gut microbiota significantly by increasing alpha diversity (observed species, ace, Shannon, and Simpson) and restoring the gut microbiota. CPE increased Firmicutes and decreased Bacteroidetes. The reduced genus Prevotella, Acinetobacter, and Morganella were positively associated with the diarrhea indices, whereas increased genus Odoribacter, Rikenella, and Roseburia were negatively associated with the diarrhea indices. The abundance of carbohydrate metabolism-related gene and SCFAs-producing bacteria were increased, which was evidenced by increased butyric acid and total SCFAs concentration in the caecum. Consequently, endocrine peptides glucagon-like peptide-1, epidermal growth factor, and peptide tyrosine tyrosine in serum were elevated. ConclusionsCPE shows a shift function on the gut microbiota in alleviating the diarrhea of mice in a dose-dependent manner. In addition, the microbial metabolites SCFAs may mediate CPE’s anti-diarrhea effect by enhancing endocrine secretion in mice.

In Vivo, Proteomic, and In Silico Investigation of Sapodilla for Therapeutic Potential in Gastrointestinal Disorders.

This study aims to delineate the effects of Manilkara zapota Linn. (Sapodilla) fruit chloroform (Mz.CHCl3) and aqueous (Mz.Aq) extracts tested through different techniques. Antidiarrheal activity and intestinal fluid accumulation were examined by using castor oil-induced diarrhea and castor oil fluid accumulation models. Isolated rabbit jejunum tissues were employed for in vitro experiments. Antimotility and antiulcer were performed through charcoal meal transient time and ethanol-induced ulcer assay, molecular studies were conducted through proteomic analysis, and virtual screening was performed by using a discovery studio visualizer (DSV). Mz.CHCl3 and Mz.Aq extracts attributed dose-dependent (50–300 mg/kg) protection (20–100%) against castor oil-induced diarrhea and dose-dependently (50–300 mg/kg) inhibited intestinal fluid secretions in mice. Mz.CHCl3 and Mz.Aq extracts produce relaxation of spontaneous and K+ (80 Mm) induced contractions in isolated tissue preparations and decreased the distance moved by charcoal in the gastrointestinal transit model in rats. It showed gastroprotective effect in ulcerative stomach of rats and decreased levels of IL-18 quantified by proteomic analysis. Histopathological results showed ethanol-induced significant gastric injury, leading to cloudy swelling, hydropic degeneration, apoptosis, and focal necrosis in all gastric zones using hematoxylin and eosin (H&E) staining. Moreover, ethanol increased the activation and the expression of tumor necrotic factor (TNF-α), cyclooxygenase (COX-2), and nuclear factor kappa-light-chain-enhancer of activated B cells (p-NFκB). In silico results were comparative to in vitro results evaluated through virtual screening. Moreover, ethanol increased the activation and expression of tumor necrotic factor, cyclooxygenase, and nuclear factor kappa-light-chain-enhancer of activated B cells. This study exhibits the gastroprotective effect of Manilkara zapota extracts in the peritoneal cavity using a proteomic and in silico approach which reveals different energy values against target proteins, which mediate the gastrointestinal functions.

Pathophysiological importance of bile cholesterol reabsorption: hepatic NPC1L1-exacerbated steatosis and decreasing VLDL-TG secretion in mice fed a high-fat diet

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide, although its pathogenesis remains to be elucidated. A recent study revealed that hepatic Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol re-absorber from bile to the liver expressed on the bile canalicular membrane, is an exacerbation factor of NAFLD. Indeed, transgenic mice with hepatic expression of human NPC1L1 under a liver-specific promoter (L1-Tg mice) developed steatosis with a high-fat diet (HFD) containing cholesterol within a few weeks. However, the mechanism underlying diet-induced hepatic NPC1L1-mediated lipid accumulation is poorly defined.MethodsTo achieve a deeper understanding of steatosis development in L1-Tg mice, the biochemical features of hepatic NPC1L1-mediated steatosis were investigated. Hemizygous L1-Tg mice and wild-type littermate controls fed a HFD or control-fat diet were used. At the indicated time points, the livers were evaluated for cholesterol and triglyceride (TG) contents as well as mRNA levels of hepatic genes involved in the maintenance of lipid homeostasis. The hepatic ability to secrete very low-density lipoprotein (VLDL)-TG was also investigated.ResultsUnlike the livers of wild-type mice that have little expression of hepatic Npc1l1, the livers of L1-Tg mice displayed time-dependent changes that indicated steatosis formation. In steatosis, there were three different stages of development: mild accumulation of hepatic cholesterol and TG (early stage), acceleration of hepatic TG accumulation (middle stage), and further accumulation of hepatic cholesterol (late stage). In the early stage, between WT and L1-Tg mice fed a HFD for 2 weeks, there were no significant differences in the hepatic expression of Pparα, Acox1, Fat/Cd36, Srebf1, and Srebf2; however, the hepatic ability to secrete VLDL-TG decreased in L1-Tg mice (P < 0.05). Furthermore, this decrease was completely prevented by administration of ezetimibe, an NPC1L1-selective inhibitor.ConclusionHepatic NPC1L1 exacerbates diet-induced steatosis, which was accompanied by decreased hepatic ability of VLDL-TG secretion. The obtained results provide a deeper understanding of L1-Tg mice as a promising NAFLD animal model that is able to re-absorb biliary-secreted cholesterol similar to humans. Furthermore, this work supports further studies of the pathophysiological impact of re-absorbed biliary cholesterol on the regulation of hepatic lipid homeostasis.

Loss of LAMTOR1 in pancreatic β‑cells increases glucose‑stimulated insulin secretion in mice

Insulin secretion from pancreatic β-cells regulates glucose metabolism and is related to various diseases including diabetes. The late endosomal/lysosomal adaptor MAPK and mTOR activator 1 (LAMTOR1) is one of the subunits of the 'Ragulator' complex and plays an important role in energy metabolism including glucose metabolism. The present study was designed to explore the role of LAMTOR1 in murine pancreatic β-cell function. A murine model with β cell-specific deficiency (βLamtor1-KO) was generated to assess β-cell function (insulin sensitivity paired with β-cell responses) by hyperglycemic clamp in vivo. Islet perfusion and mitochondrial functional analyses were performed to investigate the individual steps in the insulin secretion pathway. Results showed that glucose tolerance in vivo as well as glucose-stimulated insulin secretion in the hyperglycemic clamp and islet perfusion were higher in βLamtor1-KO mice compared to the control models. Although mitochondrial dysfunction was present, the deletion of Lamtor1 increased glutamate content in the mouse insulin granules as well as acetyl-CoA carboxylase 1 (ACC1) activity thus enhancing insulin secretion. Together, our data indicate that LAMTOR1 is important for maintaining mitochondrial function in mouse pancreatic β-cells, however deletion of Lamtor1 increases the amplification pathway induced by glutamate and ACC1, ultimately leading to increased insulin secretion. These findings suggest that knockout of Lamtor1 is a potential technique for improving pancreatic β-cell function and treating diabetes.

N-acyl taurines are endogenous lipid messengers that improve glucose homeostasis

Fatty acid amide hydrolase (FAAH) degrades 2 major classes of bioactive fatty acid amides, the N-acylethanolamines (NAEs) and N-acyl taurines (NATs), in central and peripheral tissues. A functional polymorphism in the human FAAH gene is linked to obesity and mice lacking FAAH show altered metabolic states, but whether these phenotypes are caused by elevations in NAEs or NATs is unknown. To overcome the problem of concurrent elevation of NAEs and NATs caused by genetic or pharmacological disruption of FAAH in vivo, we developed an engineered mouse model harboring a single-amino acid substitution in FAAH (S268D) that selectively disrupts NAT, but not NAE, hydrolytic activity. The FAAH-S268D mice accordingly show substantial elevations in NATs without alterations in NAE content, a unique metabolic profile that correlates with heightened insulin sensitivity and GLP-1 secretion. We also show that N-oleoyl taurine (C18:1 NAT), the most abundant NAT in human plasma, decreases food intake, improves glucose tolerance, and stimulates GPR119-dependent GLP-1 and glucagon secretion in mice. Together, these data suggest that NATs act as a class of lipid messengers that improve postprandial glucose regulation and may have potential as investigational metabolites to modify metabolic disease.

Calycosin-7-O-β-D-glucoside attenuates myocardial ischemia-reperfusion injury by activating JAK2/STAT3 signaling pathway via the regulation of IL-10 secretion in mice.

Calycosin-7-O-β-D-glucoside (CG) is the component of Astragali Radix, and the aim of the present study is to investigate whether CG protects myocardium from I/R-induced damage by the regulation of IL-10/JAK2/STAT3 signaling pathway. H9C2 cells were subjected to I/R treatment and pretreated with 1μm CG in vitro. In addition, a mouse model of myocardial I/R injury was induced by left anterior descending (LAD) coronary artery ligation and administrated with 30mg/kg CG by intravenous injection before I/R surgery. In vitro and in vivo results showed that CG up-regulated IL-10 level, activated the JAK2/STAT3 pathway, and protected myocardial cells from I/R-induced apoptosis. The hemodynamic measurement, TTC staining, TUNEL staining, and western blot results in vivo showed that the protective effects of CG on myocardial function and cell apoptosis were all reversed by the IL-10R α neutralizing antibody. CG-induced phosphorylation activation of JAK2/STAT3 signaling pathway was also suppressed by the blocking of IL-10. In summary, these findings suggest that CG might alleviate myocardial I/R injury by activating the JAK2/STAT3 signaling pathway via up-regulation of IL-10 secretion, which provides us insights into the mechanism underlying the protective effect of CG on myocardial I/R injury.

Short-chain fatty acids and regulation of pancreatic endocrine secretion in mice

ABSTRACTThe intestinal microbiota has been demonstrated to influence host metabolism, and has been proposed to affect the development of obesity and type 2 diabetes (T2D), possibly through short-chain fatty acids (SCFAs) produced by fermentation of dietary fiber. There are some indications that SCFAs inhibit glucose-stimulated insulin secretion (GSIS) in rodents, but research on this subject is sparse. However, it has been reported that receptors for SCFAs, free fatty acid receptor 2 (FFAR2) and FFAR3 are expressed not only on gut endocrine cells secreting GLP-1 and PYY, but also on pancreatic islet cells. We hypothesized that SCFAs might influence the endocrine secretion from pancreatic islets similar to their effects on the enteroendocrine cells. We studied this using isolated perfused mouse pancreas which responded adequately to changes in glucose and to infusions of arginine. None of the SCFAs, acetate, propionate and butyrate, influenced glucagon secretion, whereas they had weak inhibitory effects on somatostatin and insulin secretion. Infusions of two specific agonists of FFAR2 and FFAR3, CFMB and Compound 4, respectively, did not influence the pancreatic secretion of insulin and glucagon, whereas both induced strong increases in the secretion of somatostatin. In conclusion, the small effects of acetate, propionate and butyrate we observed here may not be physiologically relevant, but the effects of CFMB and Compound 4 on somatostatin secretion suggest that it may be possible to manipulate pancreatic secretion pharmacologically with agonists of the FFAR2 and 3 receptors, a finding which deserves further investigation.

Estradiol Enables Chronic Corticosterone to Inhibit Pulsatile Luteinizing Hormone Secretion and Suppress Kiss1 Neuronal Activation in Female Mice

Introduction: Two common responses to stress include elevated circulating glucocorticoids and impaired luteinizing hormone (LH) secretion. We have previously shown that a chronic stress level of corticosterone can impair ovarian cyclicity in intact mice by preventing follicular-phase endocrine events. Objective: This study is aimed at investigating if corticosterone can disrupt LH pulses and whether estradiol is necessary for this inhibition. Methods: Our approach was to measure LH pulses prior to and following the administration of chronic corticosterone or cholesterol in ovariectomized (OVX) mice treated with or without estradiol, as well as assess changes in arcuate kisspeptin (Kiss1) neuronal activation, as determined by co-expression with c-Fos. Results: In OVX mice, a chronic 48 h elevation in corticosterone did not alter the pulsatile pattern of LH. In contrast, corticosterone induced a robust suppression of pulsatile LH secretion in mice treated with estradiol. This suppression represented a decrease in pulse frequency without a change in amplitude. We show that the majority of arcuate Kiss1 neurons contain glucocorticoid receptor, revealing a potential site of corticosterone action. Although arcuate Kiss1 and Tac2 gene expression did not change in response to corticosterone, arcuate Kiss1 neuronal activation was significantly reduced by chronic corticosterone, but only in mice treated with estradiol. Conclusions: Collectively, these data demonstrate that chronic corticosterone inhibits LH pulse frequency and reduces Kiss1 neuronal activation in female mice, both in an estradiol-dependent manner. Our findings support the possibility that enhanced sensitivity to glucocorticoids, due to ovarian steroid milieu, may contribute to reproductive impairment associated with stress or pathophysiologic conditions of elevated glucocorticoids.

Blocking Sodium-Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice.

Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium‐taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B–mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP‐binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8 −/− mice. Stimulatory effects of NTCP inhibition were maintained in Sr‐b1 −/− mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high‐density lipoprotein–derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion.

Discovery of a novel chalcone derivative inhibiting CFTR chloride channel via AMPK activation and its anti-diarrheal application

Secretory diarrhea is one of the most common causes of death world-wide especially in children under 5 years old. Isoliquiritigenin (ISLQ), a plant-derived chalcone, has previously been shown to exert anti-secretory action in vitro and in vivo by inhibiting CFTR Cl− channels. However, its CFTR inhibition potency is considerably low (IC50 > 10 μM) with unknown mechanism of action. This study aimed to identify novel chalcone derivatives with improved potency and explore their mechanism of action. Screening of 27 chalcone derivatives identified CHAL-025 as the most potent chalcone analog that reversibly inhibited CFTR-mediated Cl− secretion in T84 cells with an IC50 of ∼1.5 μM. As analyzed by electrophysiological and biochemical analyses, the mechanism of CFTR inhibition by CHAL-025 is through AMP-activated protein kinase (AMPK), a negative regulator of CFTR activity. Furthermore, Western blot analyses and molecular dynamics (MD) results suggest that CHAL-025 activates AMPK by binding at the allosteric site of an upstream kinase calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, CHAL-025 inhibited both cholera toxin (CT) and bile acid-induced Cl− secretion in T84 cells and prevented CT-induced intestinal fluid secretion in mice. Therefore, CHAL-025 represents a promising anti-diarrheal agent that inhibits CFTR Cl− channel activity via CaMKKβ-AMPK pathways.

Role of protease-activated receptor 2 in regulation of renin synthesis and secretion in mice.

It has been reported that the serine protease kallikrein stimulates and that aprotinin, a protease inhibitor, inhibits renal renin secretion. Since direct stimulation of the protease-activated receptor (PAR) 2 increases renin secretion in isolated perfused mouse kidneys, we hypothesized that activation of PAR2 receptors by serine proteases could be involved in the synthesis and secretion of renin in vivo. We therefore determined the response of plasma renin concentration (PRC) to acute intraperitoneal administration of the PAR2 agonist SLIGRL, isoproterenol, hydralazine, furosemide, losartan, or lipopolysaccharide in conscious wild-type (WT) and Par2-deficient mice. Basal PRC was not different in Par2-deficient mice compared with WT mice. All six acute treatments caused significant increases of PRC in both WT and Par2-deficient mice. The response was significantly lower only in endotoxin-treated Par2-deficient mice. Chronic treatment with losartan, low salt intake, the combination of both, or furosemide caused an increase of PRC and renin mRNA in WT mice, whereas a high salt intake caused a decrease. Alterations in PRC and renal renin mRNA expression were not different between WT and Par2 -/- mice in response to chronic treatments. Par2-deficiency did not alter furosemide-induced diuresis and natriuresis. Systolic blood pressure responses to chronic treatments were not different between WT and Par2 -/- mice. In conclusion, deficiency of Par2 receptors does not alter renin secretion and renin gene expression modulated by a variety of typical maneuvers. However, activation of Par2 receptors by serine proteases seems to be of importance for renin secretion in the context of inflammation.

Simultaneous use of natural adjuvants and cell penetrating peptides improves HCV NS3 antigen-specific immune responses

To improve an effective hepatitis C virus (HCV) therapeutic vaccine, induction of a strong and long term HCV antigen-specific immune response is an important parameter. HCV non-structural protein 3 (NS3) has antigenic properties and plays a major role in viral clearance. In this study, DNA constructs encoding HCV NS3 and heat shock protein 27 (Hsp27)-NS3 genes, and the recombinant (r) NS3 and rHsp27-NS3 proteins complexed with HR9 and Cady-2 cell penetrating peptides (CPPs) were utilized to evaluate antibody, cytokine and Granzyme B secretion in mice. Herein, the formation of NS3 and Hsp27-NS3 DNA/ HR9 CPP complexes were revealed by gel retardation assay and protection against DNase and protease. Cady-2 peptide was used to form the nanoparticles with rNS3 and rHsp27-NS3 proteins. The size and charge of the nanoparticles were confirmed by SEM and Zetasizer instruments. Next, in vitro transfection of the nanoparticles was assessed by flow cytometry and western blotting. Finally, humoral and cellular immune responses were evaluated using different modalities in mice. Our data showed that HR9 and Cady-2 could form stable nanoparticles with DNA and proteins, respectively and enhance their delivery into HEK-293 T cells in a non-covalent approach. Furthermore, the heterologous Hsp27-NS3 DNA + HR9 prime/rHsp27-NS3+Cady-2 protein boost elicited a higher Th1 cellular immune response with a predominant IgG2a, IgG2b, IFN-γ profile and strong Granzyme B secretion than those induced by other groups. Briefly, the combination of a natural adjuvant (Hsp27) and CPPs (HR9 and Cady-2) could significantly stimulate effective immune responses as a promising approach for development of HCV therapeutic vaccines.

MiR-17-92 and miR-106b-25 clusters regulate beta cell mitotic checkpoint and insulin secretion in mice.

Adult beta cells in the pancreas are the sole source of insulin in the body. Beta cell loss or increased demand for insulin impose metabolic challenges because adult beta cells are generally quiescent and infrequently re-enter the cell division cycle. The aim of this study is to test the hypothesis that a family of proto-oncogene microRNAs that includes miR-17-92 and miR-106b-25 clusters regulates beta cell proliferation or function in the adult endocrine pancreas. To elucidate the role of miR-17-92 and miR-106b-25 clusters in beta cells, we used a conditional miR-17-92/miR-106b-25 knockout mouse model. We employed metabolic assays in vivo and ex vivo, together with advanced microscopy of pancreatic sections, bioinformatics, mass spectrometry and next generation sequencing, to examine potential targets of miR-17-92/miR-106b-25, by which they might regulate beta cell proliferation and function. We demonstrate that miR-17-92/miR-106b-25 regulate the adult beta cell mitotic checkpoint and that miR-17-92/miR-106b-25 deficiency results in reduction in beta cell mass in vivo. Furthermore, we reveal a critical role for miR-17-92/miR-106b-25 in glucose homeostasis and in controlling insulin secretion. We identify protein kinase A as a new relevant molecular pathway downstream of miR-17-92/miR-106b-25 in control of adult beta cell division and glucose homeostasis. The study contributes to the understanding of proto-oncogene miRNAs in the normal, untransformed endocrine pancreas and illustrates new genetic means for regulation of beta cell mitosis and function by non-coding RNAs. Sequencing data that support the findings of this study have been deposited in GEO with the accession code GSE126516.

Evaluation of Glutamate Dehydrogenase Activity and Insulin Secretion in Mice Exposed to Dexamethasone

Background and Aims: Diabetes is one of the most important endocrine disrupters and is associated with various hormones, including those that can lead to diabetes. Glucocorticoid use may lead to insulin resistance. Dexamethasone is one of these glucocorticoid compounds. Glutamate dehydrogenase plays a key role in the production of glutamate in the secretion of insulin. Based on these hormonal interactions, the aim of this study was to determine the activity of glutamate dehydrogenase and insulin secretion in dexamethasone-exposed mice. Materials and Methods: Twenty eight mice were divided into 4 experimental groups. Group 1 received standard normal saline as a control. Group 2 received standard food and received 1 mg / kg dexamethasone per day. Group 3 received standard diet and dexamethasone 3 mg / kg / day and group 4 with standard diet 5 mg / kg dexamethasone per day. After 21 days, the animals were killed, the pancreas and glutamate dehydrogenase, insulin, and serum glucose levels were determined. Results: Dexamethasone increased serum glucose levels significantly (P &lt;0.05). Dexamethasone increased the glutamate dehydrogenase activity and insulin levels in dexamethasone treated mice (p&lt;0.05) Conclusions: These results suggestd that dexamethasone increases glucose which leads to elevating glutamate dehydrogenase activity, and then increasing insulin. However, insulin was not enough to normalize glucose levels and led to hyperglycemia. Therefore, it is suggested to reduce dexamethasone administration.

1829-P: Acute Inhibition of Intestinal Neprilysin Enhances Glucose-Stimulated Insulin Secretion in Mice

Increased activity of the ubiquitous peptidase neprilysin in obesity and type 2 diabetes (T2D) is associated with impaired glucose homeostasis, and its inhibition has been shown to improve β-cell function. Neprilysin cleaves and inactivates insulinotropic peptides, including some produced in the intestine (e.g., GLP-1, GIP, CCK). While neprilysin is expressed in the intestine, the contribution of intestinal neprilysin to modulate β-cell function remains unknown. Thus, we sought to determine whether acute selective pharmacological inhibition of intestinal neprilysin enhances glucose-stimulated insulin secretion (GSIS) in healthy mice and could be a therapeutic target in T2D. High (20 mg/kg) or low (0.05 mg/kg) dose neprilysin inhibitor thiorphan, or vehicle, was orally administered to chow-fed C57BL/6 mice, then plasma and tissues were collected after 40 minutes to determine neprilysin activity. High dose thiorphan significantly inhibited neprilysin activity in plasma and all tissues listed below (data not shown). In contrast, low dose thiorphan inhibited neprilysin activity by 91±5% in colon, 50±12% in ileum and 62±4% in jejunum vs. vehicle (n=7-8, p&amp;lt;0.05), without altering neprilysin activity in pancreas, kidney and plasma. Thus, low dose thiorphan was used to selectively inhibit intestinal neprilysin in a separate cohort of mice 30 minutes prior to an oral glucose tolerance test (OGTT; 2g/kg glucose) in which GSIS was determined. Compared to vehicle, thiorphan significantly increased insulin levels during the OGTT (incremental AUC 0-30 minutes: 3181±606 vs. 5786±600 pmol/L.minutes-1, n=6-7, p=0.01) without altering basal insulin (95±21 vs. 114±25 pM, n=6-7, p=0.6). In summary, under physiological conditions, acute selective inhibition of intestinal neprilysin increases GSIS in vivo. Thus, intestinal neprilysin plays a role in modulating β-cell function, and strategies to inhibit neprilysin specifically in the intestine might improve β-cell dysfunction in T2D. Disclosure N. Esser: None. T.O. Mundinger: None. B. Barrow: None. S.M. Mongovin: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. Funding National Institutes of Health (DK098506); Belgian American Educational Foundation; Société Francophone du Diabète