Human Islets Of Langerhans Research Articles

A Permissive Role for Protein Kinase a in Support of Epac Agonist-Stimulated Human Islet Insulin Secretion

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2′-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was clearly detectable. Under conditions of islet perifusion, 8-pCPT-2′-O-Me-cAMP-AM (10 micromolar) potentiated 10 mM glucose-stimulated insulin secretion (GSIS), while failing to influence insulin secretion measured in the presence of 3 mM glucose. The secretagogue action of 8-pCPT-2′-O-Me-cAMP-AM was associated with K-ATP channel inhibition, depolarization and an increase of [Ca2+]i measured in single beta cells or whole islets. As expected for an Epac-selective cAMP analog, 8-pCPT-2′-O-Me-cAMP-AM (10 micromolar) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2′-O-Me-cAMP-AM (10 micromolar) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Surprisingly, treatment of human islets with an inhibitor of PKA activity (H-89, 10 micromolar), or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS; 200 micromolar), reduced GSIS measured in the absence of 8-pCPT-2′-O-Me-cAMP-AM. Furthermore, the action of 8-pCPT-2′-O-Me-cAMP-AM to potentiate GSIS was nearly abolished by H-89 and Rp-8-CPT-cAMPS. Thus, there exists a permissive role for PKA in support of glucose-stimulated and Epac-regulated human islet insulin secretion. We propose that this permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or post-priming steps of Ca2+-dependent exocytosis.

PKA-dependent potentiation of glucose-stimulated insulin secretion by Epac activator 8-pCPT-2′-O-Me-cAMP-AM in human islets of Langerhans

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2'-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM was associated with depolarization and an increase of [Ca(2+)](i) that reflected both Ca(2+) influx and intracellular Ca(2+) mobilization in islet beta-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca(2+)-dependent exocytosis.

Regulated laminin‐332 expression in human islets of Langerhans

Laminin-332 (LN-332) is a basement membrane component known to exert a beneficial effect on rat pancreatic beta cells in vitro. In this work, we analyzed the expression of LN-332 in human islets, its expression after inflammatory insults by cytokines, and the molecular mechanisms responsible for this effect. By Western blotting and RT-PCR, we showed that LN-332 was expressed in isolated human islets. By immunofluorescence on pancreas sections, we observed that labeling was confined to endocrine cells in islets. Confocal microscopy analysis on isolated islet cells revealed that labeling was granular but did not colocalize with hormone secretory granules. LN-332 was most abundant in cultured islets compared to freshly isolated islets and was found in culture medium, which suggests that it was secreted by islets. When islets were exposed to interleukin (IL)-1beta, expression and secretion of LN-332 increased as compared to control. No effect was observed with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited culture- and IL-1beta-induced LN-332 expression in islets. These results show that LN-332, known to have some beneficial effect on beta cells in vitro, is produced and secreted by endocrine islet cells and is up-regulated by stressing conditions such as culture and IL-1beta-exposure.

Block-copolymer micelles as carriers of cell signaling modulators for the inhibition of JNK in human islets of Langerhans

Here we investigate the potential of PCL-b-PEO micelles in preventing the cell death of isolated human islets of Langerhans. PCL-b-PEO micelles were loaded with c-Jun NH 2-terminal kinases inhibitor SP600125 to rescue the isolated islets. Mechanistic studies of the uptake were conducted in PC12 cells. Incorporation of SP600125 afforded 8.2 fold greater solubility of SP600125 in micelle suspension. To investigate the effectiveness of micelle-incorporated SP600125 in preventing the islet cell death, we challenged the islets with TNF-alpha, IL-1, and IFN gamma. Micelle-incorporated SP600125 did not lose its inhibitory activity during incorporation into micelles, and it protected the islets against cytokine-induced loss of viability to the same extent as control SP600125. Moreover, the concentration of micelle-incorporated SP600125 used was 13-fold lower, demonstrating the greater efficacy of micelle delivered SP600125. Micelles maintained their cytoplasmic distribution without detectable nuclear localization in islets. The inhibition of JNK was confirmed by western blots. This study suggests that micelle-based intracellular delivery of potent, poorly water soluble, cell-death-pathway inhibitors may represent a valuable addition to established delivery of cytocidal block-copolymer micelle-incorporated bioactives.

Perilipin Is Present in Islets of Langerhans and Protects against Lipotoxicity When Overexpressed in the β-Cell Line INS-1

Lipids have been shown to play a dual role in pancreatic beta-cells: a lipid-derived signal appears to be necessary for glucose-stimulated insulin secretion, whereas lipid accumulation causes impaired insulin secretion and apoptosis. The ability of the protein perilipin to regulate lipolysis prompted an investigation of the presence of perilipin in the islets of Langerhans. In this study evidence is presented for perilipin expression in rat, mouse, and human islets of Langerhans as well as the rat clonal beta-cell line INS-1. In rat and mouse islets, perilipin was verified to be present in beta-cells. To examine whether the development of lipotoxicity could be prevented by manipulating the conditions for lipid storage in the beta-cell, INS-1 cells with adenoviral-mediated overexpression of perilipin were exposed to lipotoxic conditions for 72 h. In cells exposed to palmitate, perilipin overexpression caused increased accumulation of triacylglycerols and decreased lipolysis compared with control cells. Whereas glucose-stimulated insulin secretion was retained after palmitate exposure in cells overexpressing perilipin, it was completely abolished in control beta-cells. Thus, overexpression of perilipin appears to confer protection against the development of beta-cell dysfunction after prolonged exposure to palmitate by promoting lipid storage and limiting lipolysis.

Ion channels underlying stimulus-exocytosis coupling and its cell-to-cell heterogeneity in β-cells of transplantable porcine islets of Langerhans

Given the growing interest in porcine islets as model tissue for studying the pathogenesis of human diabetes mellitus and its treatment by transplantation, we investigated stimulus-exocytosis coupling in single porcine β-cells using patch clamp electrophysiology, Ca2+ imaging, capacitance tracking and amperometry. We establish that porcine β-cells display several features prominently seen in β-cells from human islets of Langerhans. These include: (i) wide heterogeneity of electrical responsiveness to glucose; (ii) dependence of action potential activity on voltage-dependent Na+ as well as high voltage activated Ca2+ current; (iii) heterogeneity of time course of depolarization-evoked insulin granule exocytosis; and (iv) the dependence of vigorous single cell electrical activity and insulin granule exocytosis on the presence of agents that enhance cytosolic cAMP concentration. These findings promote the usefulness of porcine β-cells as a model for studying β-cell function in large mammals, including humans, as well as an appropriate source of tissue for xenotransplantation.

Isolating Human Islets of Langerhans Causes Loss of Decay Accelerating Factor (CD55) on β-Cells

It has previously been reported that human decay accelerating factor (DAF; CD55) is not expressed on cells isolated from human islets. We have investigated if this absence is caused by the islet isolation procedure and/or the single cell isolation technique. We focused on loss of DAF expression on beta-cells within the intact islet and on isolated individual beta-cells. We established that DAF was expressed in islets and on beta-cells prior to isolation by in situ analysis in the intact pancreas. In situ immunohistochemistry (IHC) was used to examine DAF expression on human pancreatic islets and isolated islets. A reverse transcriptase-polymerase chain reaction (RT-PCR) specific for human DAF mRNA was developed to measure mRNA levels in situ in islets within the intact pancreas, isolated islets, and purified beta-cells. beta-Cells were purified by fluorescence-activated cell sorting. DAF protein expression on these purified cells was measured using flow cytometry. Expression of DAF protein was present on the islets, including beta-cells within the human pancreas; however, comparative data from IHC and flow cytometry revealed the absence of DAF protein on beta-cells in both isolated islets and single cell preparations. Furthermore, compared to mRNA levels detected by in situ RT-PCR in the intact pancreas and in human HEK 293 cells, isolated islets, and purified human beta-cells showed downregulation of DAF mRNA. mRNA was detectable in both of these preparations by RT-PCR; levels were lower following both the islet isolation process (53%) and single cell preparation (a further 62%) compared to HEK 293 controls. Human islet allotransplantation might be more successful if either de novo transfer of DAF onto the isolated islets or novel techniques for islet isolation preserving DAF could be developed.

Absence of autoantibodies against glutamate decarboxylase (GAD) in the non-obese diabetic (NOD) mouse and low expression of the enzyme in mouse islets.

GAD is a major islet cell autoantigen in human type 1 diabetes mellitus. Autoantibodies are preferentially directed against the 65-kD isoform of the enzyme which is the only form expressed in human islets of Langerhans. The NOD mouse is a spontaneous model of type 1 diabetes, frequently employed in studies dealing with the immunopathogenesis of the disease. In the present study the reactivity of sera from 34 prediabetic and 15 diabetic NOD mice was tested against GAD protein present in islets of Langerhans and cerebellum, and against recombinant, semi-purified GAD-65 and GAD-67. A rabbit antiserum (K2) raised against GAD-67 could readily recognize the recombinant GAD-67 and the isoform present in rat and mouse islets and mouse brain. A MoAb (GAD-6) specific for the GAD-65 isoform reacted against the recombinant GAD-65 and the isoform present in rat islets and mouse brain, whereas no reactivity was observed when using mouse islets. However, when testing the NOD mice sera by immunohistochemistry, immunoprecipitation and Western blot, no reactivity against any of the isoforms of GAD could be detected. Using reverse transcription polymerase chain reaction (PCR), GAD-67 mRNA could be detected in mouse and rat islets and in mouse brain. GAD-65 mRNA could also be detected in rat islets and mouse brain, but apparently a much lower copy number is present in mouse islets. These findings stress important differences in the immune response occurring in the animal model NOD mouse compared with human type 1 diabetes, and emphasize that human and animal type 1 diabetes possibly represent the final outcome of several different etiological factors.

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane

Based on mouse study findings, pancreatic islet cells are supposed to lack basement membrane (BM) and interact directly with vascular endothelial BM. Until now, the BM composition of human islets has remained elusive. Immunohistochemistry with specific monoclonal and polyclonal antibodies as well as electron microscopy were used to study BM organisation and composition in human adult islets. Isolated islet cells and function-blocking monoclonal antibodies and recombinant soluble Lutheran peptide were further used to study islet cell adhesion to laminin (Lm)-511. Short-term cultures of islets were used to study Lutheran and integrin distribution. Immunohistochemistry revealed a unique organisation for human Lm-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained Lm-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the Lm alpha5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas alpha(3) and beta(1) integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in short-term culture of human islets. The adhesion of dispersed beta cells to Lm-511 was inhibited equally effectively by antibodies to integrin and alpha(3) and beta(1) subunits, and by soluble Lutheran peptide. The present results disclose a hitherto unrecognised BM organisation and adhesion mechanisms in human pancreatic islets as distinct from mouse islets.

Isolation of the Islets of Langerhans From the Human Pancreas With Magnetic Retraction

Low yield and insufficient purity limit the transplantation of human islets of Langerhans. In the rat, a new technique to isolate the islets of Langerhans was developed by intraarterial infusion of iron particles into the islet capillaries. After digestion the iron-loaded islets were purified with magnetic retraction (MR). In the present study, 10 human pancreata not suitable for clinical use were arterially injected with an iron-oxide suspension. After injection a small piece was used for histological analysis, and the tail was intraductally perfused with collagenase and manually digested. The yield was compared with 11 pancreata processed in the standard way. Nine of 10 pancreata were successfully injected with iron-oxide and digested. After MR, enrichment was achieved but the purity was not more than 50%. Similar results between the 2 groups were obtained regarding digestion times (23 ± 1.1 vs 22.7 ± 1.5 minutes) and purification yields (1749 ± 502.1 vs 2111 ± 501.8 IE/g, P = .6) for the MR vs control groups, respectively. Histological analysis revealed that 60% to 88% of the islets contained iron aggregations with substantially higher concentrations compared with the exocrine tissue. In conclusion, iron-oxide did not influence the isolation outcome before purification. Islet purification with MR gave enrichment but no pure fractions.

Function and expression of melatonin receptors on human pancreatic islets

Melatonin is known to inhibit insulin secretion from rodent beta-cells through interactions with cell-surface MT1 and/or MT2 receptors, but the function of this hormone in human islets of Langerhans is not known. In the current study, melatonin receptor expression by human islets was examined by reverse transcription-polymerase chain reaction (RT-PCR) and the effects of exogenous melatonin on intracellular calcium ([Ca2+]i) levels and islet hormone secretion were determined by single cell microfluorimetry and radioimmunoassay, respectively. RT-PCR amplifications indicated that human islets express mRNAs coding for MT1 and MT2 melatonin receptors, although MT2 mRNA expression was very low. Analysis of MT1 receptor mRNA expression at the single cell level indicated that it was expressed by human islet alpha-cells, but not by beta-cells. Exogenous melatonin stimulated increases in intracellular calcium ([Ca2+]i) in dissociated human islet cells, and stimulated glucagon secretion from perifused human islets. It also stimulated insulin secretion and this was most probably a consequence of glucagon acting in a paracrine fashion to stimulate beta-cells as the MT1 receptor was absent in beta-cells. Melatonin did not decrease 3', 5'-cyclic adenosine monophosphate (cyclic AMP) levels in human islets, but it inhibited cyclic AMP in the mouse insulinoma (MIN6) beta-cell line and it also inhibited glucose-stimulated insulin secretion from MIN6 cells. These data suggest that melatonin has direct stimulatory effects at human islet alpha-cells and that it stimulates insulin secretion as a consequence of elevated glucagon release. This study also indicates that the effects of melatonin are species-specific with primarily an inhibitory role in rodent beta-cells and a stimulatory effect in human islets.

Glucokinase activators: molecular tools for studying the physiology of insulin-secreting cells

GK (glucokinase) catalyses the phosphorylation of glucose to glucose 6-phosphate in glucosensitive cells. In pancreatic beta-cells, this reaction is the rate-limiting step of insulin release. Recent work has led to the discovery of synthetic small-molecule activators of GK that stimulate beta-cell physiology and subsequently enhance the glucose-dependent release of insulin. It is currently recognized that these compounds may represent a significant advance in the development of new agents in the treatment of diabetes. In addition, GKAs (GK activators) are emerging as reagents that are useful tools with which to probe the function of pancreatic beta-cells and other glucosensitive cells. This includes providing insights into the physiology of the beta-cell by helping to elucidate the kinetic cycle of GK, confirming the central role of glucose metabolism to the beta-cell and highlighting subtle species-dependent differences in insulin secretion between rodent and human islets of Langerhans.

A Closed System for Islet Isolation and Purification Using the COBE2991 Cell Processor May Reduce the Need of Clean Room Facilities

During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 +/- 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were then purified by adding a gradient (bottom loading). Using this closed system 1098 +/- 489 IEQ per gram pancreas were purified with a total cell viability of 67 +/- 10% and a beta-cell viability of 41 +/- 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of beta-cells was still 56 +/- 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-alpha to 40 +/- 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca2+ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn2+ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K+ (KATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na+ (TTX) and N-type Ca2+ channels (ω-conotoxin), but not L-type Ca2+ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca2+ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Glucose-Dependent Modulation of Insulin Secretion and Intracellular Calcium Ions by GKA50, a Glucokinase Activator

Because glucokinase is a metabolic sensor involved in the regulated release of insulin, we have investigated the acute actions of novel glucokinase activator compound 50 (GKA50) on islet function. Insulin secretion was determined by enzyme-linked immunosorbent assay, and microfluorimetry with fura-2 was used to examine intracellular Ca(2+) homeostasis ([Ca(2+)](i)) in isolated mouse, rat, and human islets of Langerhans and in the MIN6 insulin-secreting mouse cell line. In rodent islets and MIN6 cells, 1 micromol/l GKA50 was found to stimulate insulin secretion and raise [Ca(2+)](i) in the presence of glucose (2-10 mmol/l). Similar effects on insulin release were also seen in isolated human islets. GKA50 (1 micromol/l) caused a leftward shift in the glucose-concentration response profiles, and the half-maximal effective concentration (EC(50)) values for glucose were shifted by 3 mmol/l in rat islets and approximately 10 mmol/l in MIN6 cells. There was no significant effect of GKA50 on the maximal rates of glucose-stimulated insulin secretion. In the absence of glucose, GKA50 failed to elevate [Ca(2+)](i) (1 micromol/l GKA50) or to stimulate insulin release (30 nmol/l-10 micromol/l GKA50). At 5 mmol/l glucose, the EC(50) for GKA50 in MIN6 cells was approximately 0.3 micromol/l. Inhibition of glucokinase with mannoheptulose or 5-thioglucose selectively inhibited the action of GKA50 on insulin release but not the effects of tolbutamide. Similarly, 3-methoxyglucose prevented GKA50-induced rises in [Ca(2+)](i) but not the actions of tolbutamide. Finally, the ATP-sensitive K(+) channel agonist diazoxide (200 micromol/l) inhibited GKA50-induced insulin release and its elevation of [Ca(2+)](i.) We show that GKA50 is a glucose-like activator of beta-cell metabolism in rodent and human islets and a Ca(2+)-dependent modulator of insulin secretion.

Design and analysis of a long‐term live‐cell imaging chamber for tracking cellular dynamics within cultured human islets of Langerhans

A means of expanding islet cell mass is urgently needed to supplement the limited availability of donor islets of Langerhans for transplant. Live cell imaging of human islets in culture has the potential to identify the specific cells and processes involved in islet expansion. A novel imaging chamber was developed to facilitate long-term three-dimensional imaging of human islets during transformation. Islets have been induced to transform into duct-like epithelial cystic structures and revert back to glucose responsive endocrine cells under appropriate conditions (Jamal et al. Cell Death Differ. 2005 12:702-712). Here we aim to further our understanding by characterizing the process at a single cell level over time-essentially constructing a high resolution recorded history of each cell and its progeny during transformation and reversion. The imaging chamber enables high resolution imaging of three-dimensional islets while maintaining the structure of the islet cells and intercellular matrix components. A mathematical model was developed to validate the imaging chamber design by determining the required chamber dimensions to avoid introduction of oxygen and nutrient transport limitations. Human islets were embedded in collagen in the imaging chamber and differential interference contrast time course images were obtained at 3 min intervals. Immunofluorescent imaging confirmed that islet phenotype was maintained for at least 5 days during imaging. Analysis of the time courses confirms our ability to identify and track individual cells over time and to observe cell death and phenotype transformation in isolated human islets.

A New Method for Incorporating Functional Heparin onto the Surface of Islets of Langerhans

A novel technique is described to conjugate macromolecular heparin complexes to cell surfaces. The method is based on the dual properties of avidin-expressing binding sites for both biotin and a macromolecular complex of heparin. A quartz crystal microbalance with dissipation monitoring (QCM-D) revealed sequential binding of biotin, avidin, and heparin complexes. Large particle flow cytometry confirmed functional integrity. Confocal microscopy of the heparinized islets showed evenly distributed fluorescence. An in vitro Chandler loop model demonstrated that the biocompatibility of the new method is comparable to the previous method used on artificial materials with regard to coagulation and antithrombin uptake. The technique presented allows human islets of Langerhans to successfully be covered with functional heparin as a means to reduce instant blood-mediated inflammatory reactions induced by the innate immune system.

The Role of Arachidonic Acid and Its Metabolites in Insulin Secretion From Human Islets of Langerhans

The roles played by arachidonic acid and its cyclooxygenase (COX)-generated and lipoxygenase (LOX)-generated metabolites have been studied using rodent islets and insulin-secreting cell lines, but very little is known about COX and LOX isoform expression and the effects of modulation of arachidonic acid generation and metabolism in human islets. We have used RT-PCR to identify mRNAs for cytosolic phospholipase A(2) (cPLA(2)), COX-1, COX-2, 5-LOX, and 12-LOX in isolated human islets. COX-3 and 15-LOX were not expressed by human islets. Perifusion experiments with human islets indicated that PLA(2) inhibition inhibited glucose-stimulated insulin secretion, whereas inhibitors of COX-2 and 12-LOX enzymes enhanced basal insulin secretion and also secretory responses induced by 20 mmol/l glucose or by 50 mumol/l arachidonic acid. Inhibition of COX-1 with 100 mumol/l acetaminophen did not significantly affect glucose-stimulated insulin secretion. These data indicate that the stimulation of insulin secretion from human islets in response to arachidonic acid does not require its metabolism through COX-2 and 5-/12-LOX pathways. The products of COX-2 and LOX activities have been implicated in cytokine-mediated damage of beta-cells, so selective inhibitors of these enzymes would be expected to have a dual protective role in diabetes: they would minimize beta-cell dysfunction while maintaining insulin secretion through enhancing endogenous arachidonic acid levels.

Acylated and Unacylated Ghrelin Promote Proliferation and Inhibit Apoptosis of Pancreatic β-Cells and Human Islets: Involvement of 3′,5′-Cyclic Adenosine Monophosphate/Protein Kinase A, Extracellular Signal-Regulated Kinase 1/2, and Phosphatidyl Inositol 3-Kinase/Akt Signaling

Among its pleiotropic actions, ghrelin modulates insulin secretion and glucose metabolism. Herein we investigated the role of ghrelin in pancreatic beta-cell proliferation and apoptosis induced by serum starvation or interferon (IFN)-gamma/TNF-alpha, whose synergism is a major cause for beta-cell destruction in type I diabetes. HIT-T15 beta-cells expressed ghrelin but not ghrelin receptor (GRLN-R), which binds acylated ghrelin (AG) only. However, both unacylated ghrelin (UAG) and AG recognized common high-affinity binding sites on these cells. Either AG or UAG stimulated cell proliferation through Galpha(s) protein and prevented serum starvation- and IFN-gamma/TNF-alpha-induced apoptosis. Antighrelin antibody enhanced apoptosis in either the presence or absence of serum but not cytokines. AG and UAG even up-regulated intracellular cAMP. Blockade of adenylyl cyclase/cAMP/protein kinase A signaling prevented the ghrelin cytoprotective effect. AG and UAG also activated phosphatidyl inositol 3-kinase (PI3K)/Akt and ERK1/2, whereas PI3K and MAPK inhibitors counteracted the ghrelin antiapoptotic effect. Furthermore, AG and UAG stimulated insulin secretion from HIT-T15 cells. In INS-1E beta-cells, which express GRLN-R, AG and UAG caused proliferation and protection against apoptosis through identical signaling pathways. Noteworthy, both peptides inhibited cytokine-induced NO increase in either HIT-T15 or INS-1E cells. Finally, they induced cell survival and protection against apoptosis in human islets of Langerhans. These expressed GRLN-R but showed also UAG and AG binding sites. Our data demonstrate that AG and UAG promote survival of both beta-cells and human islets. These effects are independent of GRLN-R, are likely mediated by AG/UAG binding sites, and involve cAMP/PKA, ERK1/2, and PI3K/Akt.

Activation of the extracellular calcium-sensing receptor initiates insulin secretion from human islets of Langerhans: involvement of protein kinases

The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca(2+) homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca(2+). CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulin-dependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the beta-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between beta-cells.