Why pancreatic islets should be regarded and regulated like organs.

There are strong reasons to say that pancreatic islets are organs before they are isolated and that they should be considered to be organs once transplanted. Thus, taking into account how much we have learned about the structure and function of islet micro-organs, it seems highly illogical to on one hand consider autologous islets be regulated as organ transplants and alloislets to be regulated with the very restrictive rules used for cell transplantation. It is particularly problematic that this policy has led to restrictions that have made it next to impossible for transplants of alloislets to be carried out in the US, which is a very sad situation for the country that made so many of the advances that brought islet transplantation to the clinic.

Reanalysis of study of pancreatic effects of incretin therapy: methodological deficiencies.

A recently published study by Butler et al. concluded that incretin treatment had adverse effects on the human type 2 diabetic pancreas including 'a marked expansion of the exocrine and endocrine pancreatic compartments, the former being accompanied by increased proliferation and dysplasia and the latter by α-cell hyperplasia with the potential for evolution into neuroendocrine tumours'. Incretin therapy has become widely used for type 2 diabetes, so these conclusions have instigated major concerns with regard to patient safety. We reassessed both the clinical case information and virtual microscopy images of the same 34 cases that were used in the Butler study as well as Network for Pancreatic Organ Donation (nPOD) cases that were not included. Whereas we would like to stress that it is important to investigate in depth any indication that incretin treatment may lead to inflammation or dysplasia in the pancreas, we find that the data presented in the Butler paper have serious methodological deficiencies that preclude any meaningful conclusions.

Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells.

AIMS/HYPOTHESIS: Fetal and neonatal beta cells have poor glucose-induced insulin secretion and only gain robust glucose responsiveness several weeks after birth. We hypothesise that this unresponsiveness is due to a generalised immaturity of the metabolic pathways normally found in beta cells rather than to a specific defect. METHODS: Using laser-capture microdissection we excised beta cell-enriched cores of pancreatic islets from day 1 (P1) neonatal and young adult Sprague-Dawley rats in order to compare their gene-expression profiles using Affymetrix U34A microarrays (neonatal, n = 4; adult, n = 3). RESULTS: Using dChip software for analysis, 217 probe sets for genes/38 expressed sequence tags (ESTs) were significantly higher and 345 probe sets for genes/33 ESTs significantly lower in beta cell-enriched cores of neonatal islets compared with those of adult islets. Among the genes lower in the neonatal beta cells were key metabolic genes including mitochondrial shuttles (malate dehydrogenase, glycerol-3-phosphate dehydrogenase and glutamate oxalacetate transaminase), pyruvate carboxylase and carnitine palmitoyl transferase 2. Differential expression of these enzyme genes was confirmed by quantitative PCR on RNA from isolated neonatal (P2 until P28) and adult islets and with immunostaining of pancreas. Even by 28 days of age some of these genes were still expressed at lower levels than in adults. CONCLUSIONS/INTERPRETATION: The lack of glucose responsiveness in neonatal islets is likely to be due to a generalised immaturity of the metabolic specialisation of pancreatic beta cells.

Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells.

AIM/HYPOTHESIS: Neonatal beta cells lack glucose-stimulated insulin secretion and are thus functionally immature. We hypothesised that this lack of glucose responsiveness results from a generalised low expression of genes characteristic of mature functional beta cells. Important glucose-responsive transcription factors, Mafa and Pdx1, regulate genes involved in insulin synthesis and secretion, and have been implicated in late beta cell development. The aim of this study was to assess whether Mafa and/or Pdx1 regulates the postnatal functional maturation of beta cells. METHODS: By quantitative PCR we evaluated expression of these and other beta cell genes over the first month compared with adult. After infection with adenovirus expressing MAFA, Pdx1 or green fluorescent protein (Gfp), P2 rat islets were evaluated by RT-PCR and insulin secretion with static incubation and reverse haemolytic plaque assay (RHPA). RESULTS: At P2 most beta cell genes were expressed at about 10% of adult, but by P7 Pdx1 and Neurod1 no longer differ from adult; by contrast, Mafa expression remained significantly lower than adult through P21. Overexpression of Pdx1 increased Mafa, Neurod1, glucokinase (Gck) mRNA and insulin content but failed to enhance glucose responsiveness. Similar overexpression of MAFA resulted in increased Neurod1, Nkx6-1, Gck and Glp1r mRNAs and no change in insulin content but, importantly, acquisition of glucose-responsive insulin secretion. Both the percentage of secreting beta cells and the amount of insulin secreted per beta cell increased, approaching that of adult beta cells. CONCLUSIONS/INTERPRETATION: In the process of functional maturation acquiring glucose-responsive insulin secretion, neonatal beta cells undergo a coordinated gene expression programme in which Mafa plays a crucial role.

Dimorphic histopathology of long-standing childhood-onset diabetes.

AIMS/HYPOTHESIS: Childhood diabetes is thought to usually result from autoimmune beta cell destruction (type 1A) with eventual total loss of beta cells. Analysis of C-peptide in children characterised at diabetes onset for autoantibodies shows heterogeneous preservation of insulin secretion in long-standing diabetes. The aim of this study was to characterise the pancreases of childhood-onset diabetes in order to define the pathological basis of this heterogeneity. METHODS: We evaluated 20 cadaveric organ donor pancreases of childhood-onset long-term patients for disease heterogeneity and obtained corresponding C-peptide measurements. RESULTS: Pancreases from the majority of cadaveric donors contained only insulin-deficient islets (14 of 20). The remaining six patients (30%) had numerous insulin-positive cells within at least some islets, with two different histological patterns. Pattern A (which we would associate with type 1A diabetes) had lobular retention of areas with 'abnormal' beta cells producing the apoptosis inhibitor survivin and HLA class I. In pattern B, 100% of all islets contained normal-appearing but quantitatively reduced beta cells without survivin or HLA class I. CONCLUSIONS/INTERPRETATION: Our data demonstrate that C-peptide secretion in long-standing diabetic patients can be explained by two different patterns of beta cell survival,possibly reflecting different subsets of type 1 diabetes.

Rat islet cell aggregates are superior to islets for transplantation in microcapsules.

AIMS/HYPOTHESIS: Islet transplantation is a promising treatment for type 1 diabetes but is hampered by a shortage of donor human tissue and early failure. Research on islet cell transplantation includes finding new sources of cells and immunoisolation to protect from immune assault and tumourigenic potential. Small islet cell aggregates were studied to determine if their survival and function were superior to intact islets within microcapsules because of reduced oxygen transport limitation and inflammatory mediators. METHODS: Islet cell aggregates were generated by dispersing rat islets into single cells and allowing them to re-aggregate in culture. Rat islets and islet cell aggregates were encapsulated in barium alginate capsules and studied when cultured in low (0.5% or 2%) or normal (20%) oxygen, or transplanted into mice. RESULTS: Encapsulated islet cell aggregates were able to survive and function better than intact islets in terms of oxygen-consumption rate, nuclei counts, insulin-to-DNA ratio and glucose-stimulated insulin secretion. They also had reduced expression of pro-inflammatory genes. Islet cell aggregates showed reduced tissue necrosis in an immunodeficient transplant model and a much greater proportion of diabetic xenogeneic transplant recipients receiving islet cell aggregates (tissue volume of only 85 islet equivalents) had reversal of hyperglycaemia than recipients receiving intact islets. CONCLUSIONS/INTERPRETATION: These aggregates were superior to intact islets in terms of survival and function in low-oxygen culture and during transplantation and are likely to provide more efficient utilisation of islet tissue, a finding of importance for the future of cell therapy for diabetes.

OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas.

AIMS/HYPOTHESIS: The basic helix-loop-helix transcription factor neurogenin-3 (NGN3) commits the fates of pancreatic progenitors to endocrine cell types, but knowledge of the mechanisms regulating the choice between proliferation and differentiation of these progenitors is limited. METHODS: Using a chromatin immunoprecipitation cloning approach, we searched for direct targets of NGN3 and identified a zinc-finger transcription factor, OVO homologue-like 1 (OVOL1). Transactivation experiments were carried out to elucidate the functional role of NGN3 in Ovol1 gene expression. Embryonic and adult rodents pancreases were immunostained for OVOL1, Ki67 and NGN3. RESULTS: We showed that NGN3 negatively regulates transcription of Ovol1 in an E-box-dependent fashion. The presence of either NGN3 or NEUROD1, but not MYOD, reduced endogenous Ovol1 mRNA. OVOL1 was detected in pancreatic tissue around embryonic day 15.5, after which OVOL1 levels dramatically increased. In embryonic pancreas, OVOL1 protein levels were low in NGN3(+) or Ki67(+) cells, but high in quiescent differentiated cells. OVOL1 presence was maintained in adult pancreas, where it was detected in islets, pancreatic ducts and some acinar cells. Additionally OVOL1 presence was lacking in proliferating ductules in regenerating pancreas and induced in cells as they began to acquire their differentiated phenotype. CONCLUSIONS/INTERPRETATION: The timing of OVOL1 appearance in pancreas and its increased levels in differentiated cells suggest that OVOL1 promotes the transition of cells from a proliferating, less-differentiated state to a quiescent more-differentiated state. We conclude that OVOL1, a downstream target of NGN3, may play an important role in regulating the balance between proliferation and differentiation of pancreatic cells.

Single pancreatic beta cells co-express multiple islet hormone genes in mice.

AIMS/HYPOTHESIS: It is widely accepted that production of insulin, glucagon, somatostatin and pancreatic polypeptide in islet cells is specific to beta, alpha, delta and pancreatic polypeptide cells, respectively. We examined whether beta cells express other genes encoding islet hormones. METHODS: Nested RT-PCR was performed on single beta cells of transgenic mice with green fluorescent protein (GFP) driven by mouse insulin I promoter (MIP-GFP). RESULTS: Only 55% of adult beta cells expressed the insulin gene alone, while others expressed two or more islet hormone genes; 4% expressed all four hormone genes. In embryonic and neonatal cells, 60% to 80% of GFP(+) cells co-expressed pancreatic polypeptide and insulin genes in contrast to 29% in adult. To clarify cell fate, we conducted lineage tracing using rat insulin II promoter-cre mice crossed with reporter mice Gt(ROSA)26Sor-loxP-flanked STOP-cassette-GFP. All GFP(+) cells expressed insulin I and II genes, and showed similar heterogeneity of co-expression to that seen in MIP-GFP mice. Although we report expression of other hormone genes in a significant proportion of beta cells, our lineage tracing results demonstrate that after inducing InsII (also known as Ins2) expression, beta cell progenitors do not redifferentiate to non-beta cells. CONCLUSIONS/INTERPRETATION: This study shows co-expression of multiple hormone genes in beta cells of adult mice as well as in embryos and neonates. This finding could: (1) represent residual expression from beta cell precursors; (2) result from alternative developmental pathways for beta cells; or (3) denote the differentiation potential of these cells. It may be linked to functional heterogeneity. This heterogeneity in gene expression may provide a means to characterise the functional, cellular and developmental heterogeneity seen in beta cells.

Towards better understanding of the contributions of overwork and glucotoxicity to the beta-cell inadequacy of type 2 diabetes.

Type 2 diabetes (T2D) is characterized by reduction of beta-cell mass and dysfunctional insulin secretion. Understanding beta-cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when beta-cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, beta-cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. beta-Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains beta-cell-rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human beta-cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how beta-cell phenotype evolves as T2D develops and progresses.

Differentiation of COPAS-sorted non-endocrine pancreatic cells into insulin-positive cells in the mouse.

AIMS/HYPOTHESIS: The regenerative process in the pancreas is of particular interest, since insulin-producing beta cells are lost in diabetes. Differentiation of new beta cells from pancreatic non-endocrine cells has been reported in vivo and in vitro, a finding that implies the existence of pancreatic stem/progenitor cells. However, while tissue-specific stem cells are well documented in skin, intestine and testis, pancreatic stem cells have been elusive. We hypothesised that pancreatic stem/progenitor cells within the non-endocrine fraction could be a source of new islets in vitro. METHODS: To test if there were such cells within the pancreas, we generated pancreatic cell aggregates from tissue remaining after islet isolation from mouse insulin promoter 1-green fluorescent protein (MIP-GFP) mice. To eliminate any contamination of insulin-positive cells, we deleted all GFP-positive aggregates using COPAS Select and cultured with Matrigel. Immunohistochemistry, quantitative real-time PCR and single-cell nested RT-PCR were performed to confirm formation of insulin-producing cells. RESULTS: The GFP-negative cells were expanded as monolayers and then differentiated into three-dimensional cystic structures. After 1 week of culture, GFP-positive cells were found as clusters or single cells. By quantitative real-time PCR, no insulin mRNA was detected immediately after COPAS sorting, but after differentiation insulin mRNA of the whole preparation was 1.91 +/- 0.31% that of purified MIP-GFP beta cells. All GFP-positive cells expressed insulin 1; most expressed insulin 2, pancreas duodenum homeobox-1 and cytokeratin 19 by single cell nested RT-PCR. CONCLUSIONS/INTERPRETATION: Our data support the concept that within the exocrine (acinar and ductal) pancreas of the adult mouse there are cells that can give rise to insulin-positive cells in vitro.

Influence of diabetes on the loss of beta cell differentiation after islet transplantation in rats.

AIMS/HYPOTHESIS: Hyperglycaemia can impair beta cell function after islet transplantation. Appropriate glucose-induced insulin secretion is dependent on a unique expression pattern of genes. Here we examined the effects of diabetes on gene expression in transplanted islets. MATERIALS AND METHODS: Streptozotocin-induced diabetic or control non-diabetic Lewis rats were transplanted under the kidney capsule with an insufficient number (2,000) of syngeneic islets to normalise blood glucose levels in diabetic rats. Eighteen days after transplantation, islet grafts were retrieved and RT-PCR used to assess expression of selected genes critical for beta cell function. Islet grafts from diabetic rats transplanted with a sufficient number of islets (3,000) to normalise hyperglycaemia were used to assess the effects of correcting blood glucose levels. Additionally, gene expression of transplanted islets from non-diabetic rats was compared with freshly isolated islets. RESULTS: In islet grafts from diabetic rats, mRNA levels of several transcription factors important for the maintenance of beta cell differentiation were reduced (pancreatic and duodenal homeobox 1 [Pdx1], neurogenic differentiation 1 [Neurod1], NK6 transcription factor related, locus 1 [Nkx6.1], paired box gene 6 [Pax6]), as were genes implicated in beta cell function (Glut2 [also known as solute carrier family 2 [facilitated glucose transporter], member 2 [Slc2a2], glucokinase, insulin, islet amyloid polypeptide [Iapp]). Conversely, mRNA levels of lactate dehydrogenase, which is normally suppressed in beta cells, were increased. The majority of the changes in gene expression were normalised after correction of hyperglycaemia, indicating that the severe loss of beta cell differentiation correlates with continuous exposure to diabetes. Even islet grafts from non-diabetic rats showed a few alterations in beta cell gene expression in comparison with fresh islets. CONCLUSIONS/INTERPRETATION: Chronic hyperglycaemia contributes to the deterioration of beta cell differentiation after islet transplantation.

Changes in gene expression in beta cells after islet isolation and transplantation using laser-capture microdissection.

AIMS/HYPOTHESIS: The process of islet isolation can cause chemical and mechanical injury to beta cells. In addition, hyperglycaemia after islet transplantation can compromise beta cell function. The aim of this experiment was to evaluate changes in gene expression in endogenous islets using laser-capture microdissection (LCM). MATERIALS AND METHODS: Islets from B6AF1 mice were studied in situ in the pancreas as well as those freshly isolated or cultured for 24 h. Fresh islets were transplanted under the kidney capsule of syngeneic diabetic (streptozocin-induced) and non-diabetic mice. Frozen sections from all the samples were prepared for LCM to obtain beta cell-enriched tissue; RNA was extracted and amplified using T7 polymerase. RT-PCR was used to assess expression of selected genes critical for beta cell function (Ins, Ipf1 [previously known as Pdx1], Slc2a2 [previously known as GLUT2] and Ldha) and the stress response (Hmox1 [previously known as HO-1], Gpx1, Tnfaip3 [previously known as A20] and Fas). Immunostaining was also performed. RESULTS: In freshly isolated and cultured islets, insulin and Ipf1 mRNA levels were decreased by 40% (compared with islets in situ), while stress genes were upregulated. Comparison between in situ pancreatic islets and engrafted beta cells of cured mice showed declines in Ipf1 expression. CONCLUSIONS/INTERPRETATION: Our experiment, the first report to investigate changes in gene expression in endogenous islets using LCM, indicate that beta cells following islet isolation and residing in a foreign graft environment have decreased expression of genes involved in insulin production and increased expression of stress genes. Our data suggest that an islet graft, even in successful transplantation, may be different from endogenous islets in gene expression.

Severe hypoglycaemia post-gastric bypass requiring partial pancreatectomy: evidence for inappropriate insulin secretion and pancreatic islet hyperplasia.

AIMS/HYPOTHESIS: Postprandial hypoglycaemia following gastric bypass for obesity is considered a late manifestation of the dumping syndrome and can usually be managed with dietary modification. We investigated three patients with severe postprandial hypoglycaemia and hyperinsulinaemia unresponsive to diet, octreotide and diazoxide with the aim of elucidating the pathological mechanisms involved. METHODS: Glucose, insulin, and C-peptide were measured in the fasting and postprandial state, and insulin secretion was assessed following selective intra-arterial calcium injection. Pancreas histopathology was assessed in all three patients. RESULTS: All three patients had evidence of severe postprandial hyperinsulinaemia and hypoglycaemia. In one patient, reversal of gastric bypass was ineffective in reversing hypoglycaemia. All three patients ultimately required partial pancreatectomy for control of neuroglycopenia; pancreas pathology of all patients revealed diffuse islet hyperplasia and expansion of beta cell mass. CONCLUSIONS/INTERPRETATION: These findings suggest that gastric bypass-induced weight loss may unmask an underlying beta cell defect or contribute to pathological islet hyperplasia, perhaps via glucagon-like peptide 1-mediated pathways.

Islet transplantation outcomes in mice are better with fresh islets and exendin-4 treatment.

AIMS/HYPOTHESIS: Although islet transplantation in diabetes holds great promise, two or three donor pancreases are usually required to achieve normoglycaemia in human or rodent recipients. We investigated whether there were differences between fresh and cultured islets in terms of transplantation outcome. We also investigated the effects of normoglycaemia during engraftment and the effects of exendin-4, a glucagon-like peptide-1 receptor agonist, on islet transplantation. MATERIALS AND METHODS: Seventy-five fresh islets were transplanted to the right kidney of diabetic mice and 425 fresh islets were transplanted to the left kidney. The mice were treated with exendin-4 or vehicle for 14 days, after which the large graft was removed by left nephrectomy. In a separate set of experiments, islets cultured in the presence or absence of exendin-4 for 72 h, or fresh islets, were transplanted to diabetic mice. In both sets of experiments, blood glucose levels were monitored. RESULTS: Compared with cultured islets, fresh islets were more effective at reversing hyperglycaemia in mice. The treatment of the recipient mice with exendin-4 did not have beneficial effects on glucose homeostasis. However, when islets are cultured, exendin-4 treatment increases the rate of reversal of hyperglycaemia, but not to the degree of fresh islets. CONCLUSIONS/INTERPRETATION: Fresh islets are more effective than cultured islets at reversing hyperglycaemia. Exendin-4 has beneficial effects on islet transplantation.

Expression of the intermediate filament vimentin in proliferating duct cells as a marker of pancreatic precursor cells.

OBJECTIVES: The expression of the intermediate filament (IF) vimentin, usually considered a marker of mesenchymal cells, has been observed in the epithelial cells during embryogenesis, carcinogenesis, and dedifferentiation, suggesting that it might be useful as a marker of proliferating precursor cells in the pancreas. METHODS: Rat pancreata at E18 and at different time points after partial pancreatectomy (Px) and human and neonatal pig pancreatic tissue sections and monolayer cultured pancreatic duct cells were observed. All tissues were simultaneously immunostained with pancytokeratin and vimentin antibodies. In costained duct cells, PDX-1 or PCNA expression was also analyzed using confocal microscope images. RESULTS: In the rat embryonic pancreas at E18, all epithelial cells that formed ductlike structures expressed both cytokeratin and vimentin IF, whereas no duct cells costained for IF in the adult rat or neonatal pig pancreas. Such costaining reappeared in the following order: common pancreatic duct, main ducts, foci of regeneration and then disappeared completely at 30 days after Px. In humans, costaining was found in only 1 diabetic patient's pancreatic section, which was accompanied by massive duct cell proliferation. In monolayer culture, most of the duct cells of human and neonatal pigs coexpressed both IF proteins. Only a few costained duct cells also expressed PDX-1, and most of those cells were also stained with PCNA in rat embryonic pancreas and regenerating foci after partial Px. CONCLUSIONS: Vimentin IF expression might be a useful marker for pancreatic precursor cells and could be used to investigate the concept of the dedifferentiation of fully matured duct cells during the process of the beta-cell neogenesis.

High-fructose diet preserves beta-cell mass and prevents diabetes in nonobese diabetic mice: A potential role for increased insulin receptor substrate-2 expression.

We demonstrate that a high-fructose diet reduces the incidence of diabetes in nonobese diabetic (NOD) mice (31.2% v 57.1% on regular chow (RC); P =.009). In a second cohort of mice, we evaluated potential mechanisms for the protective effect of the high-fructose (HF) diet and whether the metabolic changes are strain-specific. Sixty NOD and 60 Balb/c mice were randomized at weaning into HF- and RC-fed groups (30 mice each) and followed for 28 weeks. Glucose tolerance testing demonstrated improved glucose tolerance in HF diet groups (P =.001 in Balb/c; P =.04 in NOD mice at 6 months). beta-cell mass was preserved in NOD mice on the HF diet, but remained unchanged in Balb/c mice. In NOD mice, hepatic insulin receptor substrate (IRS)-2 protein expression increased by 2-fold (P =.01 for 2 v 6 months) in HF-fed mice and was 53% +/- 15% higher (P =.01) in the HF diet versus RC groups at 6 months of age. IRS-2 expression was also increased in skeletal muscle of NOD mice and in both liver and muscle of Balb/c mice. Our data suggest that a HF diet improves glucose tolerance in both NOD and Balb/c mice. The improved glucose tolerance may be related to increased IRS-2 expression and, in NOD mice, preservation of beta-cell mass.

Noninvasive in vivo measurement of beta-cell mass in mouse model of diabetes.

Pancreatic beta-cell mass (BCM) is a major determinant of the quantity of insulin that can be secreted. BCM is markedly reduced in type 1 diabetes because of selective autoimmune destruction of beta-cells. Accurate assessment of BCM in human diabetes is limited to autopsy studies, which usually suffer from inadequate clinical information; thus, the development of noninvasive means of BCM measurement could be important in intervention therapy. The goal of this study was to develop such noninvasive methods for measuring BCM featuring target-specific imaging probes and to investigate whether this technique is feasible, accurate, and predictive of BCM in normal and diabetic states. Using a beta-cell-specific monoclonal antibody IC2, modified with a radioisotope chelator for nuclear imaging, we showed that highly specific binding and accumulation to beta-cells occurs after intravenous administration of the probe, with virtually no binding to exocrine pancreas or stromal tissues. Furthermore, we observed a direct correlation between accumulation of the probe with BCM in diabetic and normal animals. Nuclear imaging of the animals that received an injection of the radioactive probe showed major difference in signal intensity between normal and diabetic pancreases. The results from this study set the route for further development of imaging probes for measuring BCM that would aid in diagnosis and treatment of diabetic patients in the clinic.

Porcine neonatal pancreatic cell clusters in tissue culture: benefits of serum and immobilization in alginate hydrogel.

Porcine neonatal pancreatic cell clusters (NPCCs) may be a suitable source of insulin producing tissue for transplantation in diabetic patients. The possible beneficial effect of serum on maturation of NPCCs in vitro is difficult to achieve because of cell clumping, which can be avoided by immobilization in alginate hydrogel matrix. Collagenase treated pancreata, cultured for 4 days, formed NPCCs that were embedded in alginate cross-linked with CaCl2 and cultured in modified Ham's F10 medium with 10% fetal calf serum (FCS) for 10 days. NPCCs cultured as suspension in F10+ with 0.5% bovine serum albumin or with 10% FCS were used as control. To prevent the aggregation when cultured with serum, NPCCs were kept as a very diluted suspension. At the beginning and end of the culture, samples were taken for insulin and DNA content and immunostained for beta and non-beta cells. The culture of NPCCs immobilized in alginate resulted with 3-fold increase in insulin content and 9-fold increase in insulin/DNA ratio. Histology revealed evident increase of number of insulin- and other hormone-positive cells compared with the control. Even though 2 weeks in culture resulted in impaired glucose-induced insulin release, the amount of insulin secreted by clusters cultured in the presence of serum was 4-fold higher than in serum-free conditions. After transplantation, NPCCs retrieved from alginate reversed hyperglycemia similarly to NPCCs cultured in standard conditions. In conclusion, this study shows the feasibility of in vitro immobilization of NPCCs in alginate three-dimensional matrix, allowing cell clusters to be cultured at least two times higher density compared with culture in suspension.

Translational control is required for the unfolded protein response and in vivo glucose homeostasis.

The accumulation of unfolded protein in the endoplasmic reticulum (ER) attenuates protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) at Ser51. Subsequently, transcription of genes encoding adaptive functions including the glucose-regulated proteins is induced. We show that eIF2alpha phosphorylation is required for translation attenuation, transcriptional induction, and survival in response to ER stress. Mice with a homozygous mutation at the eIF2alpha phosphorylation site (Ser51Ala) died within 18 hr after birth due to hypoglycemia associated with defective gluconeogenesis. In addition, homozygous mutant embryos and neonates displayed a deficiency in pancreatic beta cells. The results demonstrate that regulation of translation through eIF2alpha phosphorylation is essential for the ER stress response and in vivo glucose homeostasis.