Making ß(-like)-cells from exocrine pancreas.

Creating an abundant source of ß(-like)-cells has been a major goal in diabetes research for many decades. The concept of cell plasticity has inspired many strategies towards regenerative medicine, but its successes have been limited until very recently. Today, most cell types in the pancreas are considered candidates for the generation of ß(-like)-cells through transdifferentiation. While ß(-like)-cells that are in vitro differentiated from human embryonic stem cells are already being grafted in patients, ß(-like)-cells generated by transdifferentiation are not yet ready for clinical application. These cells would however offer several advantages over the current ß(-like)-cells generated by directed differentiation, especially concerning safety issues. In addition, perfect control of the transdifferentiation efficiency would through targeted drug delivery support a non-invasive cell therapy for diabetes. Lastly, focusing on the exocrine pancreas as prime candidate makes sense in view of their abundance and high plasticity. Keeping these hopeful perspectives in mind, it is worth to continue focused research on the mechanisms that control transdifferentiation from pancreas exocrine to ß-cells.

STAT3 modulates ß-cell cycling in injured mouse pancreas and protects against DNA damage.

Partial pancreatic duct ligation (PDL) of mouse pancreas induces a doubling of the ß-cell mass mainly through proliferation of pre-existing and newly formed ß-cells. The molecular mechanism governing this process is still largely unknown. Given the inflammatory nature of PDL and inflammation-induced signaling via the signal transducer and activator of transcription 3 (STAT3), the activation and the role of STAT3 in PDL-induced ß-cell proliferation were investigated. Duct ligation stimulates the expression of several cytokines that can act as ligands inducing STAT3 signaling and phosphorylation in ß-cells. ß-Cell cycling increased by conditional ß-cell-specific Stat3 knockout and decreased by STAT3 activation through administration of interleukin-6. In addition, the level of DNA damage in ß-cells of PDL pancreas increased after deletion of Stat3. These data indicate a role for STAT3 in maintaining a steady state in the ß-cell, by modulating its cell cycle and protection from DNA damage.

Accessory cells for ß-cell transplantation.

Despite recent advances, insulin therapy remains a treatment, not a cure, for diabetes mellitus with persistent risk of glycaemic alterations and life-threatening complications. Restoration of the endogenous ß-cell mass through regeneration or transplantation offers an attractive alternative. Unfortunately, signals that drive ß-cell regeneration remain enigmatic and ß-cell replacement therapy still faces major hurdles that prevent its widespread application. Co-transplantation of accessory non-islet cells with islet cells has been shown to improve the outcome of experimental islet transplantation. This review will highlight current travails in ß-cell therapy and focuses on the potential benefits of accessory cells for islet transplantation in diabetes.

Inducible VEGF expression by human embryonic stem cell-derived mesenchymal stromal cells reduces the minimal islet mass required to reverse diabetes.

Islet transplantation has been hampered by loss of function due to poor revascularization. We hypothesize that co-transplantation of islets with human embryonic stem cell-derived mesenchymal stromal cells that conditionally overexpress VEGF (hESC-MSC:VEGF) may augment islet revascularization and reduce the minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant lentiviruses that allowed conditional (Dox-regulated) overexpression of VEGF. HESC-MSC: VEGF were characterized by tube formation assay. After co-transplantation of hESC-MSC:VEGF with murine islets in collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, we measured blood glucose, body weight, glucose tolerance and serum C-peptide. As control, islets were transplanted alone or with non-transduced hESC-MSCs. Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF. As control, 200 islets were transplanted alone. Metabolic function of islets transplanted with hESC-MSC:VEGF significantly improved, accompanied by superior graft revascularization, compared with control groups. Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone. We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice. This approach may contribute to alleviate the need for multiple donor organs per patient.

Reprogramming of human pancreatic exocrine cells to ß-like cells.

Rodent acinar cells exhibit a remarkable plasticity as they can transdifferentiate to duct-, hepatocyte- and islet ß-like cells. We evaluated whether exocrine cells from adult human pancreas can similarly respond to proendocrine stimuli. Exocrine cells from adult human pancreas were transduced directly with lentiviruses expressing activated MAPK (mitogen-activated protein kinase) and STAT3 (signal transducer and activator of transcription 3) and cultured as monolayers or as 3D structures. Expression of STAT3 and MAPK in human exocrine cells activated expression of the proendocrine factor neurogenin 3 in 50% to 80% of transduced exocrine cells. However, the number of insulin-positive cells increased only in the exocrine cells grown initially in suspension before 3D culture. Lineage tracing identified human acinar cells as the source of Ngn3- and insulin-expressing cells. Long-term engraftment into immunocompromised mice increased the efficiency of reprogramming to insulin-positive cells. Our data demonstrate that exocrine cells from human pancreas can be reprogrammed to transplantable insulin-producing cells that acquire functionality. Given the large number of exocrine cells in a donor pancreas, this approach presents a novel strategy to expand cell therapy in type 1 diabetes.

Neurogenin 3+ cells contribute to ß-cell neogenesis and proliferation in injured adult mouse pancreas.

We previously showed that injury by partial duct ligation (PDL) in adult mouse pancreas activates Neurogenin 3 (Ngn3)(+) progenitor cells that can differentiate to ß cells ex vivo. Here we evaluate the role of Ngn3(+) cells in ß cell expansion in situ. PDL not only induced doubling of the ß cell volume but also increased the total number of islets. ß cells proliferated without extended delay (the so-called 'refractory' period), their proliferation potential was highest in small islets, and 86% of the ß cell expansion was attributable to proliferation of pre-existing ß cells. At sufficiently high Ngn3 expression level, upto 14% of all ß cells and 40% of small islet ß cells derived from non-ß cells. Moreover, ß cell proliferation was blunted by a selective ablation of Ngn3(+) cells but not by conditional knockout of Ngn3 in pre-existing ß cells supporting a key role for Ngn3(+) insulin(-) cells in ß cell proliferation and expansion. We conclude that Ngn3(+) cell-dependent proliferation of pre-existing and newly-formed ß cells as well as reprogramming of non-ß cells contribute to in vivo ß cell expansion in the injured pancreas of adult mice.

Human blood outgrowth endothelial cells improve islet survival and function when co-transplanted in a mouse model of diabetes.

AIMS/HYPOTHESIS: As current islet-transplantation protocols suffer from significant graft loss and dysfunction, strategies to sustain the long-term benefits of this therapy are required. Rapid and adequate oxygen and nutrient delivery by blood vessels improves islet engraftment and function. The present report evaluated a potentially beneficial effect of adult human blood outgrowth endothelial cells (BOEC) on islet graft vascularisation and function. METHODS: Human BOEC, 5 × 10(5), were co-transplanted with a rat marginal-islet graft under the kidney capsule of hyperglycaemic NOD severe combined immunodeficiency (SCID) mice, and the effect on metabolic outcome was evaluated. RESULTS: Although vessel density remained unaffected, co-transplantation of islets with BOEC resulted in a significant and specific improvement of glycaemia and increased plasma C-peptide. Moreover, in contrast to control mice, BOEC recipients displayed reduced beta cell death and increases in body weight, beta cell proliferation and graft-vessel and beta cell volume. In vivo cell tracing demonstrated that BOEC remain at the site of transplantation and do not expand. The potential clinical applicability was underscored by the observed metabolic benefit of co-transplanting islets with BOEC derived from a type 1 diabetes patient. CONCLUSIONS/INTERPRETATION: The present data support the use of autologous BOEC in translational studies that aim to improve current islet-transplantation protocols for the treatment of brittle type 1 diabetes.

Differentiating neural crest stem cells induce proliferation of cultured rodent islet beta cells.

AIMS/HYPOTHESIS: Efficient stimulation of cycling activity in cultured beta cells would allow the design of new strategies for cell therapy in diabetes. Neural crest stem cells (NCSCs) play a role in beta cell development and maturation and increase the beta cell number in co-transplants. The mechanism behind NCSC-induced beta cell proliferation and the functional capacity of the new beta cells is not known. METHODS: We developed a new in vitro co-culture system that enables the dissection of the elements that control the cellular interactions that lead to NCSC-dependent increase in islet beta cells. RESULTS: Mouse NCSCs were cultured in vitro, first in medium that stimulated their proliferation, then under conditions that supported their differentiation. When mouse islet cells were cultured together with the NCSCs, more than 35% of the beta cells showed cycle activity. This labelling index is more than tenfold higher than control islets cultured without NCSCs. Beta cells that proliferated under these culture conditions were fully glucose responsive in terms of insulin secretion. NCSCs also induced beta cell proliferation in islets isolated from 1-year-old mice, but not in dissociated islet cells isolated from human donor pancreas tissue. To stimulate beta cell proliferation, NCSCs need to be in intimate contact with the beta cells. CONCLUSIONS/INTERPRETATION: Culture of islet cells in contact with NCSCs induces highly efficient beta cell proliferation. The reported culture system is an excellent platform for further dissection of the minimal set of factors needed to drive this process and explore its potential for translation to diabetes therapy.

Pancreatic islet and progenitor cell surface markers with cell sorting potential.

AIMS/HYPOTHESIS: The aim of the study was to identify surface bio-markers and corresponding antibody tools that can be used for the imaging and immunoisolation of the pancreatic beta cell and its progenitors. This may prove essential to obtain therapeutic grade human beta cells via stem cell differentiation. METHODS: Using bioinformatics-driven data mining, we generated a gene list encoding putative plasma membrane proteins specifically expressed at distinct stages of the developing pancreas and islet beta cells. In situ hybridisation and immunohistochemistry were used to further prioritise and identify candidates. RESULTS: In the developing pancreas seizure related 6 homologue like (SEZ6L2), low density lipoprotein receptor-related protein 11 (LRP11), dispatched homologue 2 (Drosophila) (DISP2) and solute carrier family 30 (zinc transporter), member 8 (SLC30A8) were found to be expressed in early islet cells, whereas discoidin domain receptor tyrosine kinase 1 (DDR1) and delta/notch-like EGF repeat containing (DNER) were expressed in early pancreatic progenitors. The expression pattern of DDR1 overlaps with the early pancreatic and duodenal homeobox 1 (PDX1)⁺/NK6 homeobox 1 (NKX6-1)⁺ multipotent progenitor cells from embryonic day 11, whereas DNER expression in part overlaps with neurogenin 3 (NEUROG3)⁺ cells. In the adult pancreas SEZ6L2, LRP11, DISP2 and SLC30A8, but also FXYD domain containing ion transport regulator 2 (FXYD2), tetraspanin 7 (TSPAN7) and transmembrane protein 27 (TMEM27), retain an islet-specific expression, whereas DDR1 is undetectable. In contrast, DNER is expressed at low levels in peripheral mouse and human islet cells. Re-expression of DDR1 and upregulation of DNER is observed in duct-ligated pancreas. Antibodies to DNER and DISP2 have been successfully used in cell sorting. CONCLUSIONS/INTERPRETATION: Extracellular epitopes of SEZ6L2, LRP11, DISP2, DDR1 and DNER have been identified as useful tags by applying specific antibodies to visualise pancreatic cell types at specific stages of development. Furthermore, antibodies recognising DISP2 and DNER are suitable for FACS-mediated cell purification.

Glibenclamide activates translation in rat pancreatic beta cells through calcium-dependent mTOR, PKA and MEK signalling pathways.

AIMS/HYPOTHESIS: Prolonged exposure of rat beta cells to the insulin secretagogue glibenclamide has been found to induce a sustained increase in basal insulin synthesis. This effect was calcium-dependent and localised in cells that had been degranulated by the drug. Since it was blocked by the translation inhibitor cycloheximide, we examined whether sustained exposure to glibenclamide activates translational factors by calcium-dependent signalling pathways. METHODS: Purified rat beta cells were cultured with and without glibenclamide in the presence or absence of inhibitors of calcium-dependent signalling pathways before measurement of basal and stimulated protein and insulin synthesis, and assessment of abundance of (phosphorylated) translation factors. RESULTS: A 24 h exposure to glibenclamide induced activation of four translation factors, i.e. phosphorylation of eukaryotic initiation factor (eIF) 4e binding protein 1 and ribosomal protein S6 (rpS6), and dephosphorylation of eIF-2alpha and eukaryotic elongation factor 2. The rise in phospho-rpS6 intensity was localised to a subpopulation of beta cells with low insulin content. This activation of translational factors and the associated elevation of insulin synthesis were completely blocked by the calcium channel blocker verapamil and partially blocked by the mammalian target of rapamycin (mTOR) inhibitor rapamycin, the protein kinase A (PKA) inhibitor Rp-8-Br-cAMPs and the mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase (MEK) inhibitor U0126; a combination of inhibitors exhibited additive effects. CONCLUSIONS/INTERPRETATION: Prolonged exposure to glibenclamide activates protein translation in pancreatic beta cells through the calcium-regulated mTOR, PKA and MEK signalling pathways. The observed intercellular differences in translation activation are proposed as underlying mechanism for functional heterogeneity in the pancreatic beta cell population.

Methyl succinate antagonises biguanide-induced AMPK-activation and death of pancreatic beta-cells through restoration of mitochondrial electron transfer.

BACKGROUND AND PURPOSE: Two mechanisms have been proposed to explain the insulin-sensitising properties of metformin in peripheral tissues: (a) inhibition of electron transport chain complex I, and (b) activation of the AMP activated protein kinase (AMPK). However the relationship between these mechanisms and their contribution to beta-cell death and dysfunction in vitro, are currently unclear. EXPERIMENTAL APPROACH: The effects of biguanides (metformin and phenformin) were tested on MIN6 beta-cells and primary FACS-purified rat beta-cells. Cell metabolism was assessed biochemically and by FACS analysis, and correlated with AMPK phosphorylation state and cell viability, with or without fuel substrates. KEY RESULTS: In MIN6 cells, metformin reduced mitochondrial complex I activity by up to 44% and a 25% net reduction in mitochondrial reducing potential. In rat beta-cells, metformin caused NAD(P)H accumulation above maximal glucose-inducible levels, mimicking the effect of rotenone. Drug exposure caused phosphorylation of AMPK on Thr(172) in MIN6 cell extracts, indicative of kinase activation. Methyl succinate, a complex II substrate, appeared to bypass metformin blockade of complex I. This resulted in reduced phosphorylation of AMPK, establishing a link between biguanide-induced mitochondrial inhibition and AMPK activation. Corresponding assessment of cell death indicated that methyl succinate decreased biguanide toxicity to beta-cells in vitro. CONCLUSIONS AND IMPLICATIONS: AMPK activation can partly be attributed to metformin's inhibitory action on mitochondrial complex I. Anaplerotic fuel metabolism via complex II rescued beta-cells from metformin-associated toxicity. We propose that utilisation of anaplerotic nutrients may reconcile in vitro and in vivo effects of metformin on the pancreatic beta-cell.

Ectopic expression of neurogenin 3 in neonatal pig pancreatic precursor cells induces (trans)differentiation to functional alpha cells.

AIMS/HYPOTHESIS: Neurogenin 3 (NEUROG3), a basic helix-loop-helix transcription factor that is needed for endocrine cell development in the embryonic pancreas, has been shown to induce transdifferentiation of duct cells from adult pancreas towards a neuro-endocrine phenotype. Our study explored the endocrine transdifferentiation potential of NEUROG3 in neonatal pancreatic precursor cells. MATERIALS AND METHODS: A replication-deficient adenovirus expressing Neurog3 and green fluorescent protein (GFP) (Ad-NEUROG3) was used to infect neonatal pig pancreatic cell preparations enriched for endocrine islet and cytokeratin-positive precursor cells. GFP-positive cells were sorted using flow cytometry on days 3 and 8 after infection and characterised at the transcript and protein level. For in vivo experiments, the total population of Ad-NEUROG3-infected pancreatic cells was transplanted, then later removed for determination of graft hormone content and immunohistochemistry. RESULTS: Among the GFP-positive cells, the fraction of precursor cells decreased by more than 85% at day 8 after infection, while the fraction of glucagon-positive cells increased 2.5-fold and the beta cell number remained the same. Transplantation of the Ad-NEUROG3-infected pancreatic cell preparation failed to reverse streptozotocin-induced hyperglycaemia, while non-infected cells and a control cell preparation infected with replication-deficient adenovirus expressing only GFP were able to do so. At day 109 after transplantation, kidneys grafted with Ad-NEUROG3-infected pancreatic cells contained significantly decreased insulin and increased glucagon levels. Abundant glucagon-immunopositive cells were seen in Ad-NEUROG3-infected grafts, which were virtually devoid of proliferating insulin-positive cells. CONCLUSIONS/INTERPRETATION: In summary, adenoviral delivery of NEUROG3 to pancreatic precursor cells from neonatal pig pancreas promotes alpha cell differentiation in vitro and in vivo.

Ngn3 expression during postnatal in vitro beta cell neogenesis induced by the JAK/STAT pathway.

The basic helix-loop-helix protein Neurogenin3 specifies precursor cells of the endocrine pancreas during embryonic development, and is thought to be absent postnatally. We have studied Ngn3 expression during in vitro generation of beta-cells from adult rat exocrine pancreas tissue treated with epidermal growth factor and leukaemia inhibitory factor. This treatment induced a transient expression of both Ngn3 and its upstream activator hepatocyte nuclear factor 6. Inhibition of EGF and LIF signalling by pharmacological antagonists of the JAK2/STAT3 pathway, or knockdown of Ngn3 by RNA interference prevented the generation of new insulin-positive cells. This study demonstrates that in vitro growth factor stimulation can induce recapitulation of an embryonic endocrine differentiation pathway in adult dedifferentiated exocrine cells. This could prove to be important for understanding the mechanism of beta-cell regeneration and for therapeutic ex vivo neogenesis of beta cells.

[Adult stem cells regenerate the endocrine pankreas and normalize hyperglycaemia and insulin production in diabetic mice]. / Adulte Stammzellen regenerieren das endokrine Pankreas und normalisieren Hyperglykämie und Insulinproduktion in einem Diabetesmodell der Maus.

AIMS: The potential role of adult stem cells in the regeneration of beta cells in diabetes is still controversial. Although islet cell transplantation is currently the most pursued field of research, we have investigated the capacity of multipotent adult stem cells to correct hyperglycaemia in an experimental murine diabetes model. METHODS: Cloned stem cells were labelled with eGFP or transfected with a pTie2-RFP construct to show endothelial differentiation in vivo. The beta cell toxin alloxan was injected intravenously and all mice became hyperglycaemic (> 400 mg/dl) within two days and lost more than 90 % of their beta cell mass. Stem cells were then injected either directly into the pancreas or given systemically. RESULTS: Mice that received stem cell transplantation reached normal blood glucose levels within 14 days and the beta cell mass fully recovered within one month after treatment, regaining normal body weight soon after stem cell infusion. The host pancreas then dissociated and further analysed. The eGFP+ donor cells did not express insulin and other endocrine markers, but showed a red fluorescence (RFP+) and CD31 expression instead, characteristics of endothelial cells after pTie2 activation. It was further shown that remaining (eGFP-) beta cells showed increased cell cycle activity. CONCLUSIONS: Endothelial differentiation from transplanted stem cells, induced by the environment of an injured pancreas, allows the regeneration of insulin production either through proliferation of still existing and residual beta cells in the islet or the recruitment and differentiation of beta cell progenitors mostly from the duct region via enhanced vasculogenesis and microcirculation.

[Reprogramming of adult human pancreatic duct cells as functional beta cells]. / Herprogrammering van adulte humane ductcellen van de pancreas tot functionele betacellen.

The most plausible therapy for type I diabetes is restoration of the functional beta cell mass, as is proven by the clinical success of transplantation of donor beta cells. This approach, however, is very limited by the low number of donor organs available. One solution for this shortage would be well controlled generation of beta cells for transplantation. The bona fide beta progenitor cell is not known yet. As duct cells of the adult human pancreas show a high degree of plasticity, they were genetically manipulated to transdifferentiate into beta cells. The transcription factor neurogenin 3, a master switch of embryogenesis of the endocrine pancreas, was ectopically expressed in adult human pancreatic duct cells. Neurogenin 3 induced a phenotypic shift by activation of a series of endocrine marker genes, among which insulin. However, the number of insulin-producing cells and the amount of insulin per cell were low and the transdifferentiated duct cells remained glucose insensitive. A strategy for optimizing the reprogramming of adult human duct cells to beta cells is proposed.

AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase.

We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.

AICA-riboside induces apoptosis of pancreatic beta cells through stimulation of AMP-activated protein kinase.

AIMS/HYPOTHESIS: Prolonged exposure of beta cells to low glucose concentrations triggers their apoptosis and is known to activate AMP-activated protein kinase (AMPK) in beta cell lines. We examined whether prolonged activation of AMPK can trigger apoptosis in rodent beta cells. METHODS: Primary beta cells were FACS-purified from rats, and from wild-type and AMPK(alpha2)-deficient mice. AMPK activation in beta cells was induced by the adenosine analog AICA-riboside and detected by immunoblotting using a phosphospecific antibody. Apoptosis of rodent beta cells was monitored by FACS analysis of beta cell DNA content, by direct counting of apoptotic cells using fluorescence microscopy, or by measurement of their caspase-3 activity. RESULTS: Dose-dependent and time-dependent apoptosis of the cells, concommittant with an activation of caspase-3, were suppressed by the caspase inhibitors zVAD-fmk and zDEVD-fmk. Apoptosis induction by AICA-riboside was also prevented by adding the MAPK-inhibitor SB203580 which blocked the AICA-riboside-induced phosphorylation of AMPK. Beta cells isolated from AMPK-(alpha2)-deficient mice were resistant against AICA-riboside induced apoptosis. CONCLUSION/INTERPRETATION: Sustained activation of AMPK by AICA-riboside can trigger a caspase-dependent apoptosis of pancreatic beta cells.

A comprehensive analysis of cytokine-induced and nuclear factor-kappa B-dependent genes in primary rat pancreatic beta-cells.

Type 1 diabetes mellitus results from an autoimmune destruction of pancreatic beta-cells. Cytokines, such as interleukin-1 beta and interferon-gamma, are putative mediators of immune-induced beta-cell death and, under in vitro conditions, cause beta-cell apoptosis. We have recently shown that interleukin-1 beta + interferon-gamma modifies the expression of >200 genes in beta-cells. Several of these genes are putative targets for the transcription factor nuclear factor-kappa B (NF-kappa B), and in subsequent experiments we showed that NF-kappa B activation is mostly pro-apoptotic in beta-cells. To identify cytokine-induced and NF-kappa B-regulated genes in primary rat beta-cells, we presently combined two experimental approaches: 1) blocking of NF-kappa B activation in cytokine-exposed beta-cells by a recombinant adenovirus (AdI kappa B((SA)2)) containing an inhibitor of NF-kappa B alpha (I kappa Bac) super-repressor (S32A/S36A) and 2) study of gene expression by microarray analysis. We identified 66 cytokine-modified and NF-kappa B-regulated genes in beta-cells. Cytokine-induced NF-kappa B activation decreased Pdx-1 and increased c-Myc expression. This, together with NF-kappa B-dependent inhibition of Glut-2, pro-hormone convertase-1, and Isl-1 expression, probably contributes to the loss of differentiated beta-cell functions. NF-kappa B also regulates several genes encoding for chemokines and cytokines in beta-cells. The present data suggest that NF-kappa B is a key "switch regulator" of transcription factors and gene networks controlling cytokine-induced beta-cell dysfunction and death.

Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis.

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.