Search

The pancreas originates from two epithelial evaginations of the foregut, which consist of multipotent epithelial progenitors that organize into a complex tubular epithelial network. The trunk domain of each epithelial branch consists of bipotent pancreatic progenitors (bi-PPs) that give rise to both duct and endocrine lineages, whereas the tips give rise to acinar cells.sup.1. Here we identify the extrinsic and intrinsic signalling mechanisms that coordinate the fate-determining transcriptional events underlying these lineage decisions.sup.1,2. Single-cell analysis of pancreatic bipotent pancreatic progenitors derived from human embryonic stem cells reveal that cell confinement is a prerequisite for endocrine specification, whereas spreading drives the progenitors towards a ductal fate. Mechanistic studies identify the interaction of extracellular matrix (ECM) with integrin [alpha]5 as the extracellular cue that cell-autonomously, via the F-actin-YAP1-Notch mechanosignalling axis, controls the fate of bipotent pancreatic progenitors. Whereas ECM-integrin [alpha]5 signalling promotes differentiation towards the duct lineage, endocrinogenesis is stimulated when this signalling cascade is disrupted. This cascade can be disrupted pharmacologically or genetically to convert bipotent pancreatic progenitors derived from human embryonic stem cells to hormone-producing islet cells. Our findings identify the cell-extrinsic and intrinsic mechanotransduction pathway that acts as gatekeeper in the fate decisions of bipotent pancreatic progenitors in the developing pancreas. Single-cell analysis reveals that interactions with the extracellular matrix via integrin [alpha]5 and mechanotransducer YAP1 determine whether pancreatic progenitors develop along the duct or endocrine lineages., Author(s): Anant Mamidi [sup.1] , Christy Prawiro [sup.1] , Philip A. Seymour [sup.1] , Kristian Honnens de Lichtenberg [sup.1] , Abigail Jackson [sup.1] , Palle Serup [sup.1] , Henrik Semb [...]

The pancreas is made from two distinct components: the exocrine pancreas, a reservoir of digestive enzymes, and the endocrine islets, the source of the vital metabolic hormone insulin. Human islets possess limited regenerative ability; loss of islet [beta]-cells in diseases such as type 1 diabetes requires therapeutic intervention. The leading strategy for restoration of [beta]-cell mass is through the generation and transplantation of new [beta]-cells derived from human pluripotent stem cells. Other approaches include stimulating endogenous [beta]-cell proliferation, reprogramming non-[beta]-cells to [beta]-like cells, and harvesting islets from genetically engineered animals. Together these approaches form a rich pipeline of therapeutic development for pancreatic regeneration., Author(s): Qiao Zhou [sup.1] [sup.2] , Douglas A. Melton [sup.1] [sup.2] [sup.3] Author Affiliations:(1) Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, USA(2) Harvard Stem Cell Institute, Cambridge, [...]

Although a prominent role for the brain in glucose homeostasis was proposed by scientists in the nineteenth century, research throughout most of the twentieth century focused on evidence that the function of pancreatic islets is both necessary and sufficient to explain glucose homeostasis, and that diabetes results from defects of insulin secretion, action or both. However, insulin-independent mechanisms, referred to as 'glucose effectiveness', account for roughly 50% of overall glucose disposal, and reduced glucose effectiveness also contributes importantly to diabetes pathogenesis. Although mechanisms underlying glucose effectiveness are poorly understood, growing evidence suggests that the brain can dynamically regulate this process in ways that improve or even normalize glycaemia in rodent models of diabetes. Here we present evidence of a brain-centred glucoregulatory system (BCGS) that can lower blood glucose levels via both insulin-dependent and -independent mechanisms, and propose a model in which complex and highly coordinated interactions between the BCGS and pancreatic islets promote normal glucose homeostasis. Because activation of either regulatory system can compensate for failure of the other, defects in both may be required for diabetes to develop. Consequently, therapies that target the BCGS in addition to conventional approaches based on enhancing insulin effects may have the potential to induce diabetes remission, whereas targeting just one typically does not., The escalating epidemic of obesity, metabolic syndrome and type 2 diabetes (T2D) represents one of the most pressing and costly biomedical challenges confronting modern society (1,2). However, much about the [...]

Recent advances in developmental biology have greatly expanded our understanding of progenitor cell programming and the fundamental roles that Sox9 plays in liver and pancreas organogenesis. In the last 2 [...]

We recently reported that transforming growth factor-[beta] (TGF-[beta]) induces an increase in cytosolic [Ca.sup.2+] ([[[Ca.sup.2+]].sub.cyt]) in pancreatic cancer cells, but the mechanisms by which TGF-[beta] mediates [[[Ca.sup.2+]].sub.cyt] homeostasis in these cells are currently unknown. Transient receptor potential (TRP) channels and [Na.sup.+]/[Ca.sup.2+] exchangers (NCX) are plasma membrane proteins that play prominent roles in controlling [[[Ca.sup.2+]].sub.cyt] homeostasis in normal mammalian cells, but little is known regarding their roles in the regulation of [[[Ca.sup.2+]].sub.cyt] in pancreatic cancer cells and pancreatic cancer development. Expression and function of NCX1 and TRPC1 proteins were characterized in BxPc3 pancreatic cancer cells. TGF-[beta] induced both intracellular [Ca.sup.2+] release and extracellular [Ca.sup.2+] entry in these cells; however, 2-aminoethoxydiphenyl borate [2-APB; a blocker for both inositol 1,4,5-trisphosphate ([IP.sub.3]) receptor and TRPC], La[Cl.sub.3] (a selective TRPC blocker), or KB-R7943 (a selective inhibitor for the [Ca.sup.2+] entry mode of NCX) markedly inhibited the TGF-[beta]-induced increase in [[[Ca.sup.2+]].sub.cyt]. 2-APB or KB-R7943 treatment was able to dose-dependently reverse membrane translocation of PKC[alpha] induced by TGF-[beta]. Transfection with small interfering RNA (siRNA) against NCX1 almost completely abolished NCX1 expression in BxPc3 cells and also inhibited PKC[alpha] serine phosphorylation induced by TGF-[beta]. Knockdown of NCX1 or TRPC1 by specific siRNA transfection reversed TGF-[beta]-induced pancreatic cancer cell motility. Therefore, TGF-[beta]-induces [Ca.sup.2+] entry likely via TRPC1 and NCX1 and raises [[[Ca.sup.2+]].sub.cyt] in pancreatic cancer cells, which is essential for PKC[alpha] activation and subsequent tumor cell invasion. Our data suggest that TRPC1 and NCX1 may be among the potential therapeutic targets for pancreatic cancer. transient receptor potential canonical; sodium/calcium exchanger; transforming growth factor-beta; pancreatic cancer doi: 10.1152/ajpcell.00242.2010.

Endoplasmic reticulum (ER) stress leads to accumulation of un- or misfolded proteins inside the ER and initiates the unfolded protein response (UPR). Several UPR components are physiologically involved in pancreatic development and are pathophysiologically activated during acute pancreatitis. However, the exact role of ER stress in exocrine pancreatic acini is mainly unclear. The present study examined the effects of tauroursodeoxycholic acid (TUDCA), a known ER chaperone, on acinar function and UPR components. Isolated rat pancreatic acini were stimulated by increasing concentrations of cholecystokinin (CCK-8) with or without preincubation of TUDCA. UPR components were analyzed, including chaperone binding protein (BiP), protein kinase-like ER kinase (PERK), X-box binding protein (XBP)-I, c-Jun NH2-terminal kinase (JNK), CCAAT/enhancer binding protein homologues protein (CHOP), caspase 3 activation, and apoptosis. In addition, TUDCA effects were measured on amylase secretion, calcium signaling, trypsin, and cathepsin B activation. TUDCA preincubation led to a significant increase in amylase secretion after CCK-8 stimulation, a 50% reduction of intracellular trypsin activation, and reduced cathepsin B activity, although the effects for cathepsin B were not statistical significant. Furthermore, TUDCA prevented the CCK-8-induced BiP upregulation, diminished PERK and JNK phosphorylation, and prohibited the expression of CHOP, caspase 3 activation and apoptosis. XBP-1 splicing was not altered. ER stress response mechanisms are activated in pancreatic inflammation. Chemical chaperones enhance enzyme secretion of pancreatic acini, reduce ER stress responses, and attenuate ER stress-associated apoptosis. These data hint new perspectives for an employment of chemical chaperones in the therapy of acute pancreatitis. exocrine pancreatic acini; endoplasmic reticulum stress; chemical chaperone; TUDCA doi: 10.1152/ajpgi.00423.2009.

The discovery of a signalling axis that connects nicotine responses in the brain with glucose metabolism by the pancreas sheds light on why cigarette smoking increases the risk of diabetes. A signalling axis connects nicotine responses to glucose metabolism., Author(s): Giuseppe Bruschetta, Sabrina Diano Author Affiliations: Brain-to-pancreas signalling axis links nicotine and diabetes One of the many dangers of smoking is an increased risk of diabetes, because nicotine uptake [...]

The question of whether dedicated progenitor cells exist in adult vertebrate pancreas remains controversial. Centroacinar cells and terminal duct (CA/TD) cells lie at the junction between peripheral acinar cells and the adjacent ductal epithelium, and are frequently included among cell types proposed as candidate pancreatic progenitors. However these cells have not previously been isolated in a manner that allows formal assessment of their progenitor capacities. We have found that a subset of adult CA/TD cells are characterized by high levels of ALDH1 enzymatic activity, related to high-level expression of both Aldh1a1 and Aldh1a7. This allows their isolation by FACS using a fluorogenic ALDH1 substrate. FACS-isolated CA/TD cells are relatively depleted of transcripts associated with differentiated pancreatic cell types. In contrast, they are markedly enriched for transcripts encoding Sca1, Sdf1, c-Met, Nestin, and Sox9, markers previously associated with progenitor populations in embryonic pancreas and other tissues. FACS-sorted CA/TD cells are uniquely able to form self-renewing 'pancreatospheres' in suspension culture, even when plated at clonal density. These spheres display a capacity for spontaneous endocrine and exocrine differentiation, as well as glucose-responsive insulin secretion. In addition, when injected into cultured embryonic dorsal pancreatic buds, these adult cells display a unique capacity to contribute to both the embryonic endocrine and exocrine lineages. Finally, these cells demonstrate dramatic expansion in the setting of chronic epithelial injury. These findings suggest that CA/TD cells are indeed capable of progenitor function and may contribute to the maintenance of tissue homeostasis in adult mouse pancreas. ALDH1 | CD133 | pancreatic | pancreatosphere | stem cell www.pnas.org/cgi/doi/10.1073/pnas.0912589107

An immune molecule props up cells that make insulin, thereby restoring production of the crucial hormone. An immune molecule props up cells that make insulin, thereby restoring production of the crucial hormone., Author Affiliations: Insulin-producing cells (purple cells in pale area) in the pancreas are rejuvenated by the antibody teplizumab. Credit: CNRI/Science Photo Library Light micrograph of a section through an islet [...]

Organogenesis, the process by which organs develop from individual precursor stem cells, requires that the precursor cells proliferate, differentiate, and aggregate to form a functioning structure. This process progresses through changes in 4 dimensions: time and 3 dimensions of space--4D. Experimental analysis of organogenesis, by its nature, cuts the 4D developmental process into static, 2D histological images or into molecular or cellular markers and interactions with little or no spatial dimensionality and minimal dynamics. Understanding organogenesis requires integration of the piecemeal experimental data into a running, realistic and interactive 4D simulation that allows experimentation and hypothesis testing in silico. Here, we describe a fully executable, interactive, visual model for 4D simulation of organogenic development using the mouse pancreas as a representative case. Execution of the model provided a dynamic description of pancreas development, culminating in a structure that remarkably recapitulated morphologic features seen in the embryonic pancreas. In silico mutations in key signaling molecules resulted in altered patterning of the developing pancreas that were in general agreement with in vivo data. The modeling approach described here thus typifies a useful platform for studying organogenesis as a phenomenon in 4 dimensions. 4D simulation | computational biology | pancreas | development

Exenatide is a long-acting glucagon-like peptide-1 (GLP-1) mimetic used in the treatment of type 2 diabetes. There is increasing evidence that GLP-1 can influence glycemia not only via pancreatic (insulinotropic and glucagon suppression) and gastric-emptying effects, but also via an independent mechanism mediated by portal vein receptors. The aim of our study was to investigate whether exenatide has an islet-and gastric-independent glycemia-reducing effect, similar to GLP-1. First, we administered mixed meals, with or without exenatide (20 [micro]g sc) to dogs. Second, to determine whether exenatide-induced reduction in glycemia is independent of slower gastric emptying, in the same animals we infused glucose intraportally (to simulate meal test glucose appearance) with exenatide, exenatide + the intraportal GLP-1 receptor antagonist exendin-(9-39), or saline. Exenatide markedly decreased postprandial glucose: net 0- to 135-rain area under the curve = +526 [+ or -] 315 and -536 [+ or -] 197 mg*[dl.sup.-1]*[min.sup.-1] with saline and exenatide, respectively (P < 0.05). Importantly, the decrease in plasma glucose occurred without a corresponding increase in postprandial insulin but was accompanied by delayed gastric emptying and lower glucagon. Significantly lower glycemia was induced by intraportal glucose infusion with exenatide than with saline (92 [+ or -] 1 vs. 97 [+ or -] 1 mg/dl, P < 0.001) in the absence of hyperinsulinemia or glucagon suppression. The exenatide-induced lower glycemia was partly reversed by intraportal exendin-(9-39): 95 [+ or -] 3 and 92 [+ or -] 3 mg/dl with exenatide + antagonist and exenatide, respectively (P < 0.01). Our results suggest that, similar to GLP-1, exenatide lowers glycemia via a novel mechanism independent of islet hormones and slowing of gastric emptying. We hypothesize that receptors in the portal vein, via a neural mechanism, increase glucose clearance independent of islet hormones. glucagon-like peptide-1; insulin-independent; glucose regulation

The origin of the insulin-expressing [beta]-cells in the regeneration of the adult mammalian pancreas is studied. The [beta]-cells are shown to be replaced by the duplicate existing cells in the case of normal tissue turnover and injuries.

Intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) signals are central to the mechanisms underlying fluid and protein secretion in pancreatic and parotid acinar cells. Calcium release was studied in natively buffered cells following focal laser photolysis of caged molecules. Focal photolysis of caged-inositol 1,4,5 trisphosphate (Ins[P.sub.3]) in the apical region resulted in [Ca.sup.2+] release from the apical trigger zone and, after a latent period, the initiation of an apical-to-basal [Ca.sup.2+] wave. The latency was longer and the wave speed significantly slower in pancreatic compared with parotid cells. Focal photolysis in basal regions evoked only limited [Ca.sup.2+] release at the photolysis site and never resulted in a propagating wave. Instead, an apical-to-basal wave was initiated following a latent period. Again, the latent period was significantly longer under all conditions in pancreas than parotid. Although slower in pancreas than parotid, once initiated, the apical-to-basal wave speed was constant in a particular cell type. Photo release of caged-[Ca.sup.2+] failed to evoke a propagating [Ca.sup.2+] wave in either cell type. However, the kinetics of the [Ca.sup.2+] signal evoked following photolysis of caged-Ins[P.sub.3] were significantly dampened by ryanodine in parotid but not pancreas, indicating a more prominent functional role for ryanodine receptor (RyR) following Ins[P.sub.3] receptor (Ins[P.sub.3]R) activation. These data suggest that differing expression levels of Ins[P.sub.3]R, RyR, and possibly cellular buffering capacity may contribute to the fast kinetics of [Ca.sup.2+] signals in parotid compared with pancreas. These properties may represent a specialization of the cell type to effectively stimulate [Ca.sup.2+]-dependent effectors important for the differing primary physiological role of each gland. inositol 1,4,5 trisphosphate receptors; ryanodine receptors; imaging

Cholecystokinin (CCK) has been proposed to act in a vagally dependent manner to increase pancreatic exocrine secretion via actions exclusively at peripheral vagal afferent fibers. Recent evidence, however, suggests the CCK-8s may also affect brain stem structures directly. We used an in vivo preparation with the aims of 1) investigating whether the actions of intraduodenal casein perfusion to increase pancreatic protein secretion also involved direct actions of CCK at the level of the brain stem and, if so, 2) determining whether, in the absence of vagal afferent inputs, CCK-8s applied to the dorsal vagal complex (DVC) can also modulate pancreatic exocrine secretion (PES). Sprague-Dawley rats (250-400 g) were anesthetized and the common bile-pancreatic duct was cannulated to collect PES. Both vagal deafferentation and pretreatment with the CCK-A antagonist lorglumide on the floor of the fourth ventricle decreased the casein-induced increase in PES output. CCK-8s microinjection (450 pmol) in the DVC significantly increased PES; the increase was larger when CCK-8s was injected in the left side of the DVC. Protein secretion returned to baseline levels within 30 min. Microinjection of CCK-8s increased PES (although to a lower extent) also in rats that underwent complete vagal deafferentation. These data indicate that, as well as activating peripheral vagal afferents, CCK-8s increases pancreatic exocrine secretion via an action in the DVC. Our data suggest that the CCK-8s-induced increases in PES are due mainly to a paracrine effect of CCK; however, a relevant portion of the effects of CCK is due also to an effect of the peptide on brain stem vagal circuits. brain stem; dorsal vagal complex; exocrine pancreas

Luminal nutrients are essential for the growth and maintenance of digestive tissue including the pancreas and small intestinal mucosa. Long-term loss of luminal nutrients such as during animal hibernation has been shown to result in mucosal atrophy and a corresponding stress response characterized by the induction of heat shock protein (Hsp)70 expression. This study was conducted to determine if the loss of luminal nutrients during total parenteral nutrition (TPN) would result in atrophy of the exocrine pancreas and small intestinal mucosa as well as an induction of Hsp70 expression in rats. In experiment 1, the treatment groups included an orally fed control, a saline-infused surgical control, or TPN treatment for 7 days. In experiment 2, the treatment groups included an orally fed control and TPN alone or coinfused with varying doses of glucagon-like peptide (GLP)-2, a mucosal proliferation agent, for 7 days. In experiment 1, TPN resulted in a 40% reduction in pancreatic mass that was associated with a dramatic reduction in digestive enzyme expression, enhanced apoptosis, and a 200% increase in Hsp70 expression. Conversely, heat shock cognate 70, Hsp27, and Hsp60 expression was not changed in the pancreas. In experiment 2, TPN resulted in a 30% reduction in jejunal mucosa mass and a similar induction of Hsp70 expression. The inclusion of GLP-2 during TPN attenuated jejunal mucosal atrophy and inhibited Hsp70 expression, suggesting that Hsp70 induction is sensitive to cell growth. These data indicate that pancreatic and intestinal mucosal atrophy caused by a loss of luminal nutrient stimulation is accompanied by a compensatory response involving Hsp70. total parenteral nutrition; glucagon-like peptide 2; jejunal mucosa; atrophy of the pancreas; acinar cells; heat shock protein 70

Acinar cell injury early in acute pancreatitis leads to a local inflammatory reaction and to the subsequent systemic inflammatory response, which may result in multiple organ dysfunction and death. Inflammatory mediators, including chemokines and substance P (SP), are known to play a crucial role in the pathogenesis of acute pancreatitis. It has been shown that pancreatic acinar cells produce the chemokine monocyte chemoattractant protein-1 (MCP-1) in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Similarly, SP levels in the pancreas and pancreatic acinar cell expression of neurokinin-1 receptor, the primary receptor for SP, are both increased during secretagogue-induced experimental pancreatitis. This study aims to examine the functional consequences of exposing mouse pancreatic acinar cells to SP and to determine whether it leads to proinflammatory signaling, such as production of chemokines. Exposure of mouse pancreatic acini to SP significantly increased synthesis of MCP-1, macrophage inflammatory protein-l[alpha] (MIP-l[alpha]), as well as MIP-2. Furthermore, SP also increased NF-[kappa]B activation. The stimulatory effect of SP was specific to chemokine synthesis through the NF-[kappa]B pathway, since the increase in chemokine production was completely attenuated when pancreatic acini were pretreated with the selective NF-[kappa]B inhibitor NF-[kappa]B essential modulator-binding domain peptide. This study shows that SP-induced chemokine synthesis in mouse pancreatic acinar cells is NF-[kappa]B dependent. pancreatitis; monocyte chemoattractant protein-1; macrophage inflammatory protein-1[alpha]; macrophage inflammatory protein-2; neurokinin-1 receptor

To investigate the role of transforming growth factor (TGF)-[beta] family signaling in the adult pancreas, a transgenic mouse (E-dnSmad4) was created that expresses a dominant-negative Smad4 protein driven by a fragment of the elastase promoter. Although E-dnSmad4 mice have normal growth, pancreas weight, and pancreatic exocrine and ductal histology, beginning at 4-6 wk of age, E-dnSmad4 mice show an age-dependent increase in the size of islets. In parallel, an expanded population of replicating cells expressing the E-dnSmad4 transgene is found in the stroma between the enlarged islets and pancreatic ducts. Despite the marked enlargement, E-dnSmad4 islets contain normal ratios and spatial organization of endocrine cell subtypes and have normal glucose homeostasis. Replication of cells derived from primary duct cultures of wild-type mice, but not E-dnSmad4 mice, was inhibited by the addition of TGF-[beta] family proteins, demonstrating a cell-autonomous effect of the transgene. These data show that, in the adult pancreas, TGF-[beta] family signaling plays a role in islet size by regulating the growth of a pluripotent progenitor cell residing in the periductal stroma of the pancreas. islet growth; diabetes; nestin; transgenic mice; elastase; transforming growth factor-[beta]

In isolated rat pancreatic [beta]-cells, hypotonic stimulation elicited an increase in cytosolic [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.c]) at 2.8 mM glucose. The hypotonically induced ([[Ca.sup.2+].sub.c]) elevation was significantly suppressed by nicardipine, a voltage-dependent [Ca.sup.2+] channel blocker, and by [Gd.sup.3+], amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [[Ca.sup.2+].sub.c] elevation was not inhibited by suramin, a [P.sub.2] purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of [beta]-cells and produced outwardly rectifying cation currents; [Gd.sup.3+] inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and [Gd.sup.3+] significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic [beta]-cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent [Ca.sup.2+] channels and thus elevating insulin secretion. calcium ion; swelling; patch-clamp; gadolinium

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats. isolated pancreatic acini; tachykinin receptor distribution; immunofluorescence; Western blot analysis doi:10.1152/ajpgi.00505.2005

Although ethanol abuse is the most common cause of pancreatitis, the mechanism of alcohol's effect on the pancreas is not well understood. Previously, we demonstrated that in vitro ethanol treatment of pancreatic acinar cells augmented the CCK-8-induced activation of NF-[kappa]B, a key signaling system involved in the inflammatory response of pancreatitis. In the present study, we determine the role for individual PKC isoforms in the sensitizing effect of ethanol on NF-[kappa]B activation. Dispersed rat pancreatic acini were treated with and without ethanol and then stimulated with CCK-8; 100 nM CCK-8 caused both NF-[kappa]B and PKC-[delta], -[epsilon], and -[zeta] activation, whereas 0.1 nM CCK-8 did not increase PKC-[epsilon], PKC-[zeta], or NF-[kappa]B activity. CCK-8 (0.1 nM) did activate PKC-[delta]. PKC-[epsilon] activator alone did not cause NF-[kappa]B activation; however, together with 0.1 nM CCK-8, it caused NF-[kappa]B activation. Ethanol activated PKC-[epsilon] without affecting other PKC isoforms or NF-[kappa]B activity. Of note, stimulation of acini with ethanol and 0.1 nM CCK-8 resulted in the activation of PKC-[delta], PKC-[epsilon], and NF-[kappa]B. The NF-[kappa]B activation to 0.1 nM CCK-8 in ethanol-pretreated acini was inhibited by both PKC-[delta] inhibitor and PKC-[epsilon] inhibitor. Taken together, these results demonstrate the different modes of activation of PKC isoforms and NF-[kappa]B in acini stimulated with ethanol, high-dose CCK-8, and low-dose CCK-8, and furthermore suggest that activation of both PKC-[epsilon] and -[delta] is required for NF-[kappa]B activation. These results suggest that ethanol enhances the CCK-8-induced NF-[kappa]B activation at least in part through its effects on PKC-[epsilon]. alcoholic pancreatitis; cholecystokinin; inflammatory response; protein kinase C-[epsilon] translocation activator doi:10.1152/ajpgi.00579.2005

The pancreas is an elongated gland, light tan or pinkish in color, nestled alongside the small intestine and adjacent to the stomach. The organ is composed of two functionally separate [...]

We have developed a method to visualize fluorescent protein-labeled [beta]-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the [beta]-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, [beta]-cell clusters, and single [beta]-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary [beta]-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red [beta]-cells. The imaging technique that we have developed, coupled with the transgenic mice in which [beta]-cells and [beta]-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states. development of pancreas; islet formation

ATP-sensitive potassium ([K.sub.ATP]) channels of pancreatic [beta]-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane [K.sub.ATP] channels determines the sensitivity of [beta]-cells to glucose stimulation. The [K.sub.ATP] channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of [K.sub.ATP] channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of [K.sub.ATP] channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic [beta]-cell line, INS-1, that express endogenous [K.sub.ATP] channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of [beta]-cell [K.sub.ATP] channels. endoplasmic reticulum-associated degradation; sulfonylurea receptor-1 Kir6.2; proteasome inhibitors

We investigated the pre- and postsynaptic effects of pancreatic polypeptide (PP) on identified pancreas-projecting neurons of the rat dorsal motor nucleus of the vagus in thin brain stem slices. Perfusion with PP induced a Trx- and apamin-sensitive, concentration-dependent outward (22% of neurons) or inward current (21% of neurons) that was accompanied by a decrease in input resistance; PP was also found to affect the amplitude of the action potential afterhyperpolarization. The remaining 57% of neurons were unaffected. PP induced a concentration-dependent inhibition in amplitude of excitatory (n = 22 of 30 neurons) and inhibitory (n = 13 of 17 neurons) postsynaptic currents evoked by electrical stimulation of the adjacent nucleus of the solitary tract, with an estimated [EC.sub.50] of 30 nM for both. The inhibition was accompanied by an alteration in the paired pulse ratio, suggesting a presynaptic site of action. PP also decreased the frequency, but not amplitude, of spontaneous excitatory (n = 6 of 11 neurons) and inhibitory currents (n = 7 of 9 neurons). In five neurons, chemical stimulation of the area postrema (AP) induced a TTX-sensitive inward (n = 3) or biphasic (outward and inward) current (n = 2). Superfusion with PP reversibly reduced the amplitude of these chemically stimulated currents. Regardless of the PP-induced effect, the vast majority of responsive neurons had a multipolar somata morphology with dendrites projecting to areas other than the fourth ventricle or the central canal. These results suggest that pancreas-projecting rat dorsal motor nucleus of the vagus neurons are heterogeneous with respect to their response to PP, which may underlie functional differences in the vagal modulation of pancreatic functions. brain stem; parasympathetic; gastrointestinal

Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential ([V.sub.m]) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Forster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoyl phosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from [V.sub.m] -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in [V.sub.m]. When [V.sub.m] was measured simultaneously with intracellular [Ca.sup.2+], all active cells showed tight coupling of [V.sub.m] to islet cell [Ca.sup.2+] changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans. pancreatic [beta]-cell; optical electrophysiology; islet electrical coupling

Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas. Keywords: Morpholino knockdown; Degenerate PCR; Epithelial precursor; Development; Exocrine pancreas; Endocrine pancreas

Although NF-[kappa]B plays an important role in pancreatitis, mechanisms underlying its activation remain unclear. We investigated the signaling pathways mediating NF-[kappa]B activation in pancreatic acinar cells induced by high-dose cholecystokinin-8 (CCK-8), which causes pancreatitis in rodent models, and TNF-[alpha], which contributes to inflammatory responses of pancreatitis, especially the role of PKC isoforms. We determined subcellular distribution and kinase activities of PKC isoforms and NF-[kappa]B activation in dispersed rat pancreatic acini. We applied isoform-specific, cell-permeable peptide inhibitors to assess the role of individual PKC isoforms in NF-[kappa]B activation. Both CCK-8 and TNF-[alpha] activated the novel isoforms PKC-[delta] and -[epsilon] and the atypical isoform PKC-[zeta] but not the conventional isoform PKC-[alpha]. Inhibition of the novel PKC isoforms but not the conventional or the atypical isoform resulted in the prevention of NF-[kappa]B activation induced by CCK-8 and TNF-[alpha]. NF-[kappa]B activation by CCK-8 and TNF-[alpha] required translocation but not tyrosine phosphorylation of PKC-[delta]. Activation of PKC-[delta], PKC-[epsilon], and NF-[kappa]B with CCK-8 involved both phosphatidylinositol-specific PLC and phosphatidylcholine (PC)-specific PLC, whereas with TNF-[alpha] they only required PC-specific PLC for activation. Results indicate that CCK-8 and TNF-[alpha] initiate NF-[kappa]B activation by different PLC pathways that converge at the novel PKCs ([delta] and [epsilon]) to mediate NF-[kappa]B activation in pancreatic acinar cells. These findings suggest a key role for the novel PKCs in pancreatitis. phosphatidylcholine-specific phospholipase C; phosphatidylinositol-specific phospholipase C: Src kinases; translocation; tyrosine phosphorylation

The pancreatic islet is a functional microorgan involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet [beta] cells through an increase in redox state, which can be measured by NAD(P)H autofluorescence. Glucose concentrations over [approximately equal to] 7 mM generate synchronous oscillations in [beta] cell intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]]i), which lead to pulsatile insulin secretion. Prevailing models assume that the pancreatic islet acts as a functional syncytium, and the whole islet [[Ca.sup.2+]]i response has been modeled in terms of islet bursting and pacemaker models. To test these models, we developed a microfluidic device capable of partially stimulating an islet, while allowing observation of the NAD(P)H and [[Ca.sup.2+]]i responses. We show that [beta] cell [[Ca.sup.2+]]i oscillations occur only within regions stimulated with more than [approximately equal to] 6.6 mM glucose. Furthermore, we show that tolbutamide, an antagonist of the ATP-sensitive [K.sup.+] channel, allows these oscillations to travel farther into the nonstimulated regions of the islet. Our approach shows that the extent of [Ca.sup.2+] propagation across the islet depends on a delicate interaction between the degree of coupling and the extent of ATP-sensitive [K.sup.+]-channel activation and illustrates an experimental paradigm that will have utility for many other biological systems. [beta] cell | ATP-sensitive [K.sup.+] channels | NAD(P)H

Pancreatic [beta] cells play a central role in maintaining glucose homeostasis because they secrete insulin in response to increased level of blood glucose; failure of this capacity constitutes a major component of the pathogenesis of diabetes. The identification of key regulators of pancreatic [beta]-cell differentiation is relevant for the overall understanding of this process and for future experiments aimed at regenerating insulin-producing [beta] cells from pancreatic or embryonic stem cells. Several studies using transgenic or knockout mice have established that the development and function of pancreatic [beta] cells are controlled by several genes encoding specific transcription factors. By inactivating the homeobox gene Pax4, we previously demonstrated that its function is required for the formation of mature insulin-producing cells. Here, we show that during pancreas ontogeny, Pax4 is expressed in differentiating endocrine cells, including cells. Pax4 activity appears essential for appropriate initiation of [beta]-cell differentiation because loss of Pax4 prevents the expression of Pdx1, HB9 and insulin in [beta]-cell precursors. This role of Pax4 appears to be accomplished via its genetic interaction with another homeobox gene, Nkx2.2. Keywords: Pax4; Nkx2.2; Pax6; Mouse; Pancreas; [beta]-cell differentiation

Dietary regulation of digestive enzyme secretion from the pancreas is essential for the breakdown of macronutrients in the gastrointestinal tract. [Ca.sub.2+]-responsive heat stable protein (CRHSP)-28 is a regulatory protein that modulates the exocytosis of digestive enzymes from pancreatic acinar cells. In the present study, isoelectric focusing and immunoblotting were used to characterize CRHSP-28 phosphorylation in isolated rat acinar cells and also after hormonal and dietary stimulation of rat pancreas in vivo. CRHSP-28 was highly phosphorylated in isolated acini after stimulation with a physiologic range of concentrations of cholecystokinin-octapeptide (CCK-8). Activation of the high affinity state of the CCK-A receptor with the synthetic peptide JMV-180 confirmed the physiologic relevance of the response. CRHSP-28 phosphorylation was contingent on elevated cellular [Ca.sub.2+] because it was maximally stimulated by [Ca.sub.2+] ionophore, but unchanged after protein kinase C, cAMP or cyclic guanosine monophosphate activation. Intravenous infusion of rats with a secretory concentration of the CCK analog, caerulein, stimulated CRHSP-28 phosphorylation by 100% over control (P < 0.01) within 15 min of dosing. Moreover, CRHSP-28 phosphorylation was stimulated by 150% over control (P < 0.05) immediately after consumption of a semipurified AIN-93 diet. These data demonstrate that CRHSP-28 phosphorylation occurs in vivo and can be used as a functional indicator of nutrientdriven acinar cell activation. J. Nutr. 133: 3072-3075, 2003. KEY WORDS: * exocrine pancreas* phosphorylation * CRHSP-28* secretion * [Ca.sup.2+]-signaling

In some tissues, amino acids (AA) stimulate translation initiation via interactions between eukaryote initiation factor (elF) 4E-binding protein 1 (4E-BP1), elF4E and elF4G. Dietary AA have been shown to induce pancreatic proteases independently of cholecystokinin in rats, the mechanism of which has not yet been clarified. In the present study, we examined the mechanism in rats for protease induction by dietary AA and determined the involvement of translation initiation. Male Wistar/ST rats were fed a 20 or 60% casein or AA mixture diet for 7 d and were intravenously injected with [[sup.35]S] methionine (Met) 30 min before killing on d 7 (expt. 1). In expt. 2, rats were fed a 20 or 60% AA diet for 7 d and after food deprivation and refeeding with the respective diet on d 7 were killed at 0, 1 or 3 h. We measured mRNA and [[sup.35]S] Met incorporation into chymotrypsinogen, phosphorylation status of 4E-BP1 and the association of elF4E with 4E-BP1 or elF4G. In expt. 1, chymotrypsin activity and synthesis were higher in both of the 60% diet groups than in the 20% diet groups, but the mRNA level and 4E-BP1 status did not differ. In expt. 2, chymotrypsin activity increased in the 60% AA diet group in a time-dependent manner. The translation initiation activity via the mTOR pathway indicated an increase similar to chymotrypsin activity. There were no differences in chymotrypsin mRNA level at any point. These results indicate that dietary AA induce chymotrypsin synthesis by promoting translation, and transient activation of translation initiation via mTOR may be associated with this induction. J. Nutr. 133: 3052-3057, 2003. KEY WORDS: * dietary amino acids * chymotrypsin * translation initiation * eukaryote initiation factor * rats

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase [A.sub.2] (cPL[A.sub.2][alpha]) and its lipolytic products arachidonic acid and lysophosphatidylcholine on [Ca.sup.2+]-dependent exocytosis in single mouse pancreatic [beta]-cells. cPL[A.sub.2][alpha] dose dependently (E[C.sub.50] = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell [Ca.sup.2+] current or cytoplasmic [Ca.sup.2+] levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPL[A.sub.2][alpha]-stimulated exocytosis was antagonized by the specific cPL[A.sub.2][alpha] inhibitor AACOC[F.sub.3]. [Ca.sup.2+]-evoked exocytosis was reduced by 40% in cells treated with AACOC[F.sub.3] or an antisense oligonucleotide against cPL[A.sub.2][alpha]. The action of cPL[A.sub.2][alpha] was mimicked by a combination of arachidonic acid and lysophosphatidyl-choline (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve [Cl.sup.-] uptake through ClC-3 [Cl.sup.-] channels. Accordingly, the stimulatory action of cPL[A.sub.2][alpha] was inhibited by the [Cl.sup.-] channel inhibitor DIDS and in cells pretreated with ClC-3 [Cl.sup.-] channel antisense oligonucleotides. We propose that cPL[A.sub.2][alpha] has an important role in controlling the rate of exocytosis in [beta]-cells. This effect of cPL[A.sub.2][alpha] reflects an enhanced transgranular [Cl.sup.-] flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine. arachidonic acid; ClC-3 chloride channels; insulin exocytosis; lysophospholipids; phospholipase [A.sub.2]

[member of]-Polylysine ([member of]-PL) has been used as a food additive in Japan for many years. In this study, it inhibited human and porcine pancreatic lipase activity in substrate emulsions containing bile salts and phosphatidylcholine, in the concentration range of 10-1000 mg/L. At the same concentrations, it also destroyed the emulsifying activity, suggesting that lipase inhibitory activity and emulsion breakdown activity were associated. [member of]-PL inhibited porcine pancreatic lipase activity and destroyed emulsion breakdown activity at 1000 mg/L in the substrate containing bile salts and phosphatidylcholine alone. [member of]-PL did not inhibit lipase activity or affect emulsifying activity at 1000 mg/L in the substrates containing arabic gum and polyvinyl alcohol. A comparison of lipase inhibitory activity between [member of]-PL and three types of [alpha]-PL with differing polymerization rates was performed. The lipase inhibitory activity of [member of]-PL was not different from that of [alpha]-PL (44 lysine residues). [member of]-PL maintained its inhibitory activity after incubation with trypsin, [alpha]-chymotrypsin and pepsin, whereas [alpha]-PL did not. The effect of [member of]-PL on postprandial hypertriacylglyceridemia was investigated in rats. The plasma triacylglycerol concentration in rats intragastrically administered [greater than or equal to] 15 mg/kg of both fat emulsion and [member of]-PL was significantly lower at 2 and 3 h after administration than that in rats administered fat emulsion alone (P < 0.05). These results strongly suggest that [member of]-PL is able to suppress dietary fat absorption from the small intestine by inhibiting pancreatic lipase activity. J. Nutr. 133: 1887-1891, 2003. KEY WORDS: * [member of]-polylysine * lipase inhibitory activity * emulsion breakdown activity * hypotriacylglyceridemic activity * rats

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice ([GLP-1R.sup.-/-] and [GIPR.sup.-/-], respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in [GIPR.sup.-/-] than in wild-type ([GIPR.sup.+/+]) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in [GIPR.sup.-/-] mice compared with [GIPR.sup.+/+] mice. Paradoxically, immunocytochemical studies showed a significant increase in [beta]-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, [GIPR.sup.-/-] mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression. insulin; GLP-1; pancreas perfusion; cAMP; incretin

Glucagon-like peptide (GLP) 1 is produced through posttranslational processing of proglucagon and acts as a regulator of various homeostatic events. Among its analogs, however, the function of GLP-1-(1-37), synthesized in small amounts in the pancreas, has been unclear. Here, we find that GLP-1-(1-37) induces insulin production in developing and, to a lesser extent, adult intestinal epithelial cells in vitro and in vivo, a process mediated by up-regulation of the Notch-related gene ngn3 and its downstream targets, which are involved in pancreatic endocrine differentiation. These cells became responsive to glucose challenge in vitro and reverse insulin-dependent diabetes after implantation into diabetic mice. Our findings suggest that efficient induction of insulin production in intestinal epithelial cells by GLP-1-(1-37) could represent a new therapeutic approach to diabetes mellitus.

Pancreatic duodenal homeobox-1 (Pdx1) is a transcription factor, and its phosphorylation is thought to be essential for activation of insulin gene expression. This phosphorylation is related to a concomitant shift in molecular mass from 31 to 46 kDa. However, we found that Pdx1 was modified by SUMO-1 (small ubiquitin-related modifier 1) in [beta]-TC-6 and COS-7 cells, which were transfected with Pdx1 cDNA. This modification contributed to the increase in molecular mass of Pdx1 from 31 to 46 kDa. Additionally, sumoylated Pdx1 localized in the nucleus. The reduction of SUMO-1 protein by use of RNA interference (SUMO-iRNAs) resulted in a significant decrease in Pdx1 protein in the nucleus. A 34-kDa form of Pdx1 was detected by the cells exposed to SUMO-iRNAs in the presence of lactacystin, a proteasome inhibitor. Furthermore, the reduced nuclear sumoylated Pdx1 content was associated with significant lower transcriptional activity of the insulin gene. These findings indicate that SUMO-1 modification is associated with both the localization and stability of Pdx1 as well as its effect on insulin gene activation. small ubiquitin-related modifier 1; pancreatic duodenal homeobox-1; ribonucleic acid interference

Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic [alpha] cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor ([Gcgr.sup.-/-]). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. [Gcgr.sup.-/-] mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to [alpha] cell, and to a lesser extent, [delta] cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. [Gcgr.sup.-/-] mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and [alpha] and [delta] cell numbers. Furthermore, the lean phenotype of [Gcgr.sup.-/-] mice suggests glucagon action may be involved in the regulation of whole body composition.

Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin in vitro. We show that constitutive expression of Pax4 ([Pax4.sup.+]), and to a lesser extent Pdx1 ([Pdx1.sup.+]), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestin-expressing (nestin+) cells also increases. Constitutive Pax4 expression combined with selection of nestin + cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult [beta] cells. In response to glucose, [Pax4.sup.+] and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.

In view of the recent success in pancreatic islet transplantation, interest in treating diabetes by the delivery of insulin-producing [beta]-cells has been renewed. Because differentiated pancreatic [beta]-cells cannot be expanded significantly in vitro, [beta]-cell stem or progenitor cells are seen as a potential source for the preparation of transplantable insulin-producing tissue. In addition to embryonic stem (ES) cells, several potential adult islet/[beta]-cell progenitors, derived from pancreas, liver, and bone marrow, are being studied. To date, none of the candidate cells has been fully characterized or is clinically applicable, but pancreatic physiology makes the existence of one or more types of adult islet stem cells very likely. It also seems possible that pluripotential stem cells, derived from the bone marrow, contribute to adult islet neogenesis. In future studies, more stringent criteria should be met to clonally define adult islet/[beta]-cell progenitor cells. If this can be achieved, the utilization of these cells for the generation of insulin-producing [beta]-cells in vitro seems to be feasible in the near future. pancreatic islets; liver oval cells; embryonic stem cells; nestin; insulin; islet-like clusters; [beta]-cell; transplantation

Herein, the function of pancreatic metallothionein (MT)-I during zinc deficiency in pregnancy was examined using transgenic mice, which constitutively express the mouse MT-I gene driven by the rat elastase I promoter. Pancreatic MT protein levels and zinc levels were elevated significantly in the transgenic mice compared with those in control mice. Pregnant transgenic and control mice were fed zinc-deficient (1 [micro]g/g beginning at d 8) or zinc-adequate (50 [micro]g/g) diets during pregnancy, and the effects on the morphology of embryos were determined at d 14 of pregnancy (d 1 = vaginal plug). As other indicators of zinc deficiency, maternal pancreatic MT levels, as well as the expression of zinc-regulated genes in the embryonic visceral yolk sac were examined. Under these experimental conditions of moderate dietary zinc deficiency, 21.3% of the embryos in control mice exhibited morphological defects, whereas only 5.8% of the embryos in the elastase-MT-I transgenic females had developed abnormally by d 14. Surprisingly, dietary zinc deficiency caused a >95% decrease in pancreatic MT protein concentration in these transgenic mice. This suggests the post-transcriptional control of MT protein levels during zinc deficiency because the rat elastase I promoter is not metal-regulated. The decrease in pancreatic MT protein levels was paralleled by a dramatic decrease in the relative abundance of MT-I mRNA and a dramatic increase in the relative abundance of the zinc/iron regulated transporter-related zinc transporter-4 (ZIP4) mRNA in the embryonic visceral yolk sac. Thus, the constitutive overexpression of pancreatic MT-I in these mice attenuated, but did not prevent the effects of maternal or embryonic zinc deficiency under these conditions. Overall, these findings are consistent with the hypothesis that mouse pancreatic MT-I may participate in providing a labile pool of maternal zinc for the developing embryo during periods of zinc deficiency. KEY WORDS: * mice * metallothionein * pregnancy * transgenic * pancreas * zinc homeostasis