Different combinations of GABA, BMP7, and Activin A induced the in vitro differentiation of rat pancreatic ductal stem cells into insulin-secreting islet-like cell clusters.

AIMS: We investigated the in vitro differentiation of adult rat PDESCs into ß-like cells through supplementation of different combinations of GABA, BMP7, and Activin A in basic culture media. MATERIALS AND METHODS: The PDESCs were cultured using different inducement combinations for 28 days and microscopy, dithizone (DTZ) staining, immunohistochemical staining, real-time PCR, and glucose-stimulated insulin secretion (GSIS) assay were used to delineate the differentiation inducement potential of these combinations. KEY FINDINGS: The results show that after 28 days, the PDESCs were differentiated into ICCs containing insulin-secreting ß-like cells in different groups treated with A + B, A + G, B + G, and A + B + G but not in the control group. Upon DTZ staining the cells in ICCs were stained crimson red, demonstrating the presence of ß-like cells in ICCs and the immunohistochemistry showed the expression of Pdx1 and insulin in ICCs. Further, on 28 d the expression of Pdx1 and insulin mRNA was high in inducement groups as compared to the control group and ß-like cells in ICCs also secreted insulin and C-peptide upon glucose stimulation. Thus, the supplementation of GABA, BMP7, and Activin A in different combinations in basic culture media can induce the in vitro differentiation of PDESCs into ICCs containing ß-like cells. SIGNIFICANCE: The in vitro development of ß-like cells is a herald for cell therapy of diabetic patients and our results are a step closer towards finding the cure for diabetes.

Copper promotes sheep pancreatic duct organoid growth by activation of an antioxidant protein 1-dependent MEK-ERK pathway.

Proper amounts of copper supplemented in livestock feed improve the physical growth and traits of farm animals. The pancreas is an important organ with both exocrine and endocrine portions. To investigate the role and mechanism of copper in the sheep pancreas, we first established sheep pancreatic duct organoids (sPDOs). We found that an appropriate amount of copper benefited the formation and growth of sPDOs, whereas excess or deficient copper damaged sPDOs. We found that the proliferation-stimulating effect of copper was related to the copper chaperone antioxidant protein 1 (ATOX1)-dependent activation of MEK-ERK1/2 signaling. Atox1 knockdown suppressed the cell proliferation of sPDOs, even in the presence of the MEK activator. These results indicate that moderate concentrations of copper promote sPDO growth through ATOX1-regulated cell proliferation by activation of MEK-ERK. Moreover, our study indicates that organoids may be a useful model to study organ growth mechanisms in livestock.

The MiR-135b-BMAL1-YY1 loop disturbs pancreatic clockwork to promote tumourigenesis and chemoresistance.

Circadian disruption has been implicated in tumour development, but the underlying mechanism remains unclear. Here, we show that the molecular clockwork within malignant human pancreatic epithelium is disrupted and that this disruption is mediated by miR-135b-induced BMAL1 repression. miR-135b directly targets the BMAL1 3'-UTR and thereby disturbs the pancreatic oscillator, and the downregulation of miR-135b is essential for the realignment of the cellular clock. Asynchrony between miR-135b and BMAL1 expression impairs the local circadian gating control of tumour suppression and significantly promotes tumourigenesis and resistance to gemcitabine in pancreatic cancer (PC) cells, as demonstrated by bioinformatics analyses of public PC data sets and in vitro and in vivo functional studies. Moreover, we found that YY1 transcriptionally activated miR-135b and formed a 'miR-135b-BMAL1-YY1' loop, which holds significant predictive and prognostic value for patients with PC. Thus, our work has identified a novel signalling loop that mediates pancreatic clock disruption as an important mechanism of PC progression and chemoresistance.

CFTR expression but not Cl- transport is involved in the stimulatory effect of bile acids on apical Cl-/HCO3- exchange activity in human pancreatic duct cells.

OBJECTIVES: Low doses of chenodeoxycholate (CDC) stimulate apical anion exchange and HCO3(-) secretion in guinea pig pancreatic duct cells (Gut. 2008;57:1102-1112). We examined the effects of CDC on intracellular pH (pHi), intracellular Ca(2+) concentration ([Ca(2+)]i), and apical Cl(-)/HCO3(-) exchange activity in human pancreatic duct cells and determined whether any effects were dependent on cystic fibrosis transmembrane conductance regulator (CFTR) expression and Cl(-) channel activity. METHODS: Polarized CFPAC-1 cells (expressing F508del CFTR) were transduced with Sendai virus constructs containing complementary DNAs for either wild-type CFTR or beta-galactosidase. Microfluorimetry was used to record pHi and [Ca(2+)]i and apical Cl(-)/HCO3(-) exchange activity. Patch clamp experiments were performed on isolated guinea pig duct cells. RESULTS: Chenodeoxycholate induced a dose-dependent intracellular acidification and a marked increase in [Ca(2+)]i in CFPAC-1 cells. CFTR expression slightly reduced the rate of acidification but did not affect the [Ca(2+)]i changes. Luminal administration of 0.1 mmol/L of CDC significantly elevated apical Cl(-)/HCO3(-) exchange activity but only in cells that expressed CFTR. However, CDC did not activate CFTR Cl(-) conductance. CONCLUSIONS: Bile salts modulate pHi, [Ca(2+)]i, and apical anion exchange activity in human pancreatic duct cells. The stimulatory effect of CDC on anion exchangers requires CFTR expression but not CFTR channel activity.

Effects of bile acids on pancreatic ductal bicarbonate secretion in guinea pig.

BACKGROUND AND AIMS: Acute pancreatitis is associated with significant morbidity and mortality. Bile reflux into the pancreas is a common cause of acute pancreatitis and, although the bile can reach both acinar and ductal cells, most research to date has focused on the acinar cells. The aim of the present study was to investigate the effects of bile acids on HCO(3)(-) secretion from the ductal epithelium. METHODS: Isolated guinea pig intralobular/interlobular pancreatic ducts were microperfused and the effects of unconjugated chenodeoxycholate (CDC) and conjugated glycochenodeoxycholate (GCDC) on intracellular calcium concentration ([Ca(2+)](i)) and pH (pH(i)) were measured using fluorescent dyes. Changes of pH(i) were used to calculate the rates of acid/base transport across the duct cell membranes. RESULTS: Luminal administration of a low dose of CDC (0.1 mM) stimulated ductal HCO(3)(-) secretion, which was blocked by luminal H(2)DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). In contrast, both luminal and basolateral administration of a high dose of CDC (1 mM) strongly inhibited HCO(3)(-) secretion. Both CDC and GCDC elevated [Ca(2+)](i), and this effect was blocked by BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid), caffeine, xestospongin C and the phospholipase C inhibitor U73122. BAPTA-AM also inhibited the stimulatory effect of low doses of CDC on HCO(3)(-) secretion, but did not modulate the inhibitory effect of high doses of CDC. CONCLUSIONS: It is concluded that the HCO(3)(-) secretion stimulated by low concentrations of bile acids acts to protect the pancreas against toxic bile, whereas inhibition of HCO(3)(-) secretion by high concentrations of bile acids may contribute to the progression of acute pancreatitis.

Pineapple juice labeled with gadolinium: a convenient oral contrast for magnetic resonance cholangiopancreatography.

The aim of our study was to prepare in vitro a pineapple juice (PJ) solution labeled with a minimal gadolinium concentration working as a negative contrast agent in heavily T2-weighted imaging and to assess that solution in vivo as a negative oral contrast agent for magnetic resonance cholangiopancreatography (MRCP). Three PJs were compared in vitro according to their T2. Increasing concentrations of gadolinium (Gd)-DOTA in PJ were assessed in vitro for T2 reduction. Single-shot turbo spin echo T2-weighted MR cholangiopancreatograms were obtained for 35 patients with suspected biliopancreatic duct disease, before and after ingestion of the PJ/Gd-DOTA solution. Signal intensity (SI) measurements of gastroduodenal lumens, pancreatobiliary ducts, and image quality scores were obtained systematically before and after contrast ingestion. The in vitro selected Gd-DOTA concentration in the PJ was 2.76 mmol/l. Ingestion of 180 ml of PJ labeled with 1 ml of Gd-DOTA eliminated efficiently the gastroduodenal SI in MRCP, improving significantly the rates of complete visualization of the pancreatobiliary ducts (P<0.01) and the MRCP image quality scores (P<0.05). All patients easily ingested the contrast solution and found the solution palatable. PJ labeled with gadolinium constituted an efficient and convenient negative oral contrast agent for MRCP.

Neonatal pig pancreatic duct-derived insulin-producing cells: preliminary in vitro studies.

Neonatal pig pancreata could represent an ideal tissue resource for donor islets for transplantation trials. Because functional islet beta-cells could derive from precursors situated in the ductal system, and neonatal animals are better suitable than adults for recovering such elements, we have examined whether isolated neonatal pancreatic ducts (NPD) could form insulin-producing cells. NPD, retrieved from the pancreas by collagenase digestion, were cultured for 2 weeks. A compact tissue monolayer detached by trypsin was re-incubated to form upon culture. The primary tissue monolayer was plated, yielding secondary monolayers that were supplemented in culture with the following factors: insulin transferrin selenium, niacinamide, keratinocyte growth factor, and high glucose, which promoted formation of islet cell-like clusters during 30 days of culture. Upon reaching 50 to 100 microm in diameter, the cell clusters were subjected to morphologic examination (assessment of viability by staining with ethidium bromide+fluorescein diacetate [EB+FD]; staining for insulin with diphenylthiocarbazone [DTZ]); DNA assay; insulin radioimmunoassay both in the basal state and after in vitro static incubation with high glucose; immunolabeling with anti-insulin fluorescent antibodies. Of the cell clusters, 80% were composed of viable cells that faintly showed DTZ staining. Basal insulin was 16.7 microU/mL, but no insulin response was elicited by stimulation with high glucose. Acid-ethanol extraction showed high insulin levels in the clusters. Finally, immunofluorescence for insulin was positive, indicating the presence of beta-cell-like committed elements. In conclusion, NPD may differentiate into insulin-producing cells, which are at a very early stage when the glucose-sensing apparatus is still immature.

Low-protein diet during early life causes a reduction in the frequency of cells immunopositive for nestin and CD34 in both pancreatic ducts and islets in the rat.

Feeding a low-protein (LP) diet to pregnant and lactating rats impairs pancreatic islet mass and insulin release in the offspring, leading to glucose intolerance as adults. We hypothesized that an LP diet changes the number of pancreatic endocrine precursor cells or cells supporting endocrine cell neogenesis. Pregnant rats were given LP (8% protein) or a control (20% protein) diet from conception until postnatal d 21. Cells containing nestin, CD34, or c-Kit were quantified in pancreata of the offspring. Stellate cells immunoreactive for nestin were seen to be adjacent to ductal epithelium and were resident within the islets. These were proliferative and immunonegative for cytokeratin 20, fibronectin, tyrosine hydroxylase, pancreatic duodenal homeobox 1, Nk homeodomain transcription factor 6.1, or insulin, but expressed vimentin. Approximately 20% of islet nestin-positive cells also expressed the endothelial cell marker platelet endothelial cell adhesion molecule-1. Both ducts and islets also contained CD34- and c-Kit-positive cells with similar morphology to those expressing nestin. Offspring from rats fed the LP diet had significantly less nestin/CD34-positive cells and reduced expression of nestin mRNA. Within islets, there was an associated decrease in cell proliferation and in cells immunopositive for pancreatic duodenal homeobox 1. Nestin-positive cell number within islets correlated positively with the percent area of beta-cells. Supplementation of pregnant and lactating rats with taurine reversed the deficits in mean islet area and nestin-positive cells caused by the LP diet within the islets of the offspring. Nutritional programming of postnatal beta-cell mass may involve an altered abundance of cells expressing nestin and/or CD34, which may limit endocrine cell development.

Effect of Bak Foong pills on exocrine pancreatic-bile secretion.

We have recently demonstrated that Bak Foong Pills (BFP), a well-known Chinese medicine widely used for treating gynecological disorders, stimulates human colonic epithelial anion secretion, which was mediated by intracellular cAMP and Ca(2+). The present study further investigated the effect of BFP on exocrine pancreatic-bile secretion using in vivo and in vitro approaches. Duodenal infusion of BFP ethanol extract (1 g/kg) in rats produced increases in the volume and protein output of pancreatic-bile juice, but did not affect its pH. Surgical ablation of vagal neural pathway slightly reduced the effect of BFP on the protein output and volume, indicating that the vagal nerve pathway was not the major player in medicating the effect of BFP on exocrine pancreatic-bile secretion. Using CAPAN-1 cell line, a human pancreatic duct cell line, in conjunction with the short-circuit current (I(SC)) measurements, we further demonstrated that BFP could directly stimulate pancreatic HCO(3)(-) secretion. Basolateral addition of BFP (600 microg/ml) produced averaged charges transported of 2100+/-382.5 microC/cm(2), which was blocked by apical addition of Cl(-) channel blocker. Removal of HCO(3)(-) from the Krebs-Henseleit (K-H) solution inhibited the BFP-induced I(SC) by more than 95%. The present results suggest that BFP could improve digestive function by stimulating pancreatic protein and HCO(3)(-) secretion.

Bak Foong Pills stimulate anion secretion across normal and cystic fibrosis pancreatic duct epithelia.

The present study examined the effect of Bak Foong Pills (BFP), an over-the-counter traditional Chinese medicine (China registration no. Z980035), on anion secretion and the underlying signaling pathways in normal and cystic fibrosis pancreatic duct cell lines, CAPAN-1 and CFPAC-1, respectively, using the short-circuit current technique. Apical addition of BFP ethanol extract (600 microg/ml) induced a fast transient I(SC) peak that was followed by a slower but more sustained increase in I(SC) in CAPAN-1 cells. However, the response to BFP in CFPAC-1 was predominantly the first transient peak. Apical addition of DIDS (200 microM) inhibited the first peak by more than 60% in both cell lines without significantly affecting the second I(SC) rise. More than 85% of the BFP-induced first transient in both cell lines was inhibited when extra and intracellular Ca(2+) was chelated or emptied by pre-treatment with BAPTA (100 microM) and thapsigargin (10 microM), respectively. Acute addition of PMA (1 microM), a PKC activator, blocked more than 95% of the BFP-induced first peak in both cell lines, consistent with previously reported PKC modulation of Ca(2+)-dependent pancreatic anion secretion. The BFP-induced second I(SC) rise in CAPAN-1 could be inhibited by 73.6% and 71.13% by pretreatment of the cells with MDL-12330A (20 microM), an adenylate cyclase inhibitor and Rp-cAMP (200 microM), a cyclic AMP antagonist, respectively. However, less than 25% of the I(SC) was inhibited by combined treatment with BAPTA and thapsigargin. The second rise was also completely blocked by DPC (2mM) or Glibenclamide (1mM). The results indicate that BFP ethanol extract stimulates pancreatic duct anion secretion in normal and CF cells via different signaling pathways involving both Ca(2+) and cAMP.

Calcium-activated chloride currents and non-selective cation channels in a novel cystic fibrosis-derived human pancreatic duct cell line.

The modulation of ion fluxes across the plasma membrane of epithelial cells is central for fluid secretion and absorption. Their disruption can lead to pathological states. An example is cystic fibrosis (CF), a disease characterized by abnormal functioning of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-modulated chloride channel. Here we report the characterization of calcium-activated, DIDS sensitive chloride current and non-selective calcium-activated cation channels in a novel human pancreatic duct cell line (YHV-1) derived from a non-delta F508 mutation CF patient bearing a severe phenotype. Southern blot analysis of the CFTR gene indicates a distinct electrophoretic pattern for the region spanned by exons 15-24, a result presumably related to a mutation which has yet to be identified. In contrast to large calcium-activated chloride currents there were no cAMP-dependent CFTR-type chloride currents. Non-selective cation channels were blocked by intracellular ATP and activated by intracellular calcium and cAMP. We propose the cell line YHV-1 as a suitable model for studying pancreatic ion and fluid secretion alterations in CF.

Isolation and culture of rhesus monkey pancreatic ductules and ductule-like epithelium.

The objective of this work was to devise methods for the isolation and culture of duct epithelium from rhesus monkey pancreas with the expectation that such methods would be applicable to the human pancreas. This objective is important because of the role duct epithelium appears to play in human diseases such as pancreatic cancer and cystic fibrosis. Pieces of freshly procured pancreas were minced and enzymatically dissociated, resulting in a digest that contained a few isolated ductules (intralobular ducts) as well as numerous small tissue fragments consisting of roughly equal proportions of ductular and acinar cells. These fragments were suspended in a rat tail collagen gel and cultured for up to 2 weeks in a medium supplemented with cholera toxin, epidermal growth factor, and other additives. A few cystic ductular fragments were initially observed among a large number of predominantly solid fragments. Later, most of the solid fragments also became cystic and eventually resembled the ductules except for being spherical. Autoradiographic analysis of DNA synthesis showed that the cysts possessed a proliferative potential. The cysts consisted almost entirely of ductule-like epithelium with no recognizable acinar cells, and exhibited greatly reduced concentrations of the acinar marker enzymes amylase, chymotrypsin, and gamma-glutamyl transferase. In contrast, the specific activity of the duct marker enzyme carbonic anhydrase was elevated in freshly isolated digests compared with the whole pancreas and this elevated activity was maintained for 4-5 days of culture, after which it declined. Other evidence for the ductular nature of the cysts was their low density relative to freshly isolated acinar tissue, their ability to distend (suggestive of fluid/electrolyte secretion), and the accumulation of mucins at the apical borders of the cells. The results show that fragments of rhesus monkey pancreas that are enriched in ductular epithelium assume some of the properties of ductular cells when cultured in a collagen gel. These epithelial preparations should facilitate biochemical and physiological studies of this important pancreatic cell type.