Emerging diabetes therapies: Bringing back the ß-cells.

BACKGROUND: Stem cell therapies are finally coming of age as a viable alternative to pancreatic islet transplantation for the treatment of insulin-dependent diabetes. Several clinical trials using human embryonic stem cell (hESC)-derived ß-like cells are currently underway, with encouraging preliminary results. Remaining challenges notwithstanding, these strategies are widely expected to reduce our reliance on human isolated islets for transplantation procedures, making cell therapies available to millions of diabetic patients. At the same time, advances in our understanding of pancreatic cell plasticity and the molecular mechanisms behind ß-cell replication and regeneration have spawned a multitude of translational efforts aimed at inducing ß-cell replenishment in situ through pharmacological means, thus circumventing the need for transplantation. SCOPE OF REVIEW: We discuss here the current state of the art in hESC transplantation, as well as the parallel quest to discover agents capable of either preserving the residual mass of ß-cells or inducing their proliferation, transdifferentiation or differentiation from progenitor cells. MAJOR CONCLUSIONS: Stem cell-based replacement therapies in the mold of islet transplantation are already around the corner, but a permanent cure for type 1 diabetes will likely require the endogenous regeneration of ß-cells aided by interventions to restore the immune balance. The promise of current research avenues and a strong pipeline of clinical trials designed to tackle these challenges bode well for the realization of this goal.

Cellular Therapies and Regenerative Strategies for Diabetes - Proceeding of the STEMSO Conference.

Diabetes is now considered a growing global epidemic with sizable negative effects on patients' quality of life and life expectancy, and escalating economic impact (41% growth in the past five year), now representing health care ex-penditure impact of 256 Billion/year in the US alone. Objectives of cellular therapies and regenerative medicine strategies for treatment of diabetes are to reverse the disease condition and prevent the development of the severe chronic complications that can affect most organ systems in a large proportion of patients over time. Cell based therapies include the combination of immunomodulatory approaches aimed at restoring self tolerance (i.e., in the case of autoimmune diabetes) and at inducing permanent acceptance of transplanted tissues (in the case of allogeneic donors), or immune protection (i.e., engineered microenvironment and/or encapsulation) so that the immune system can no longer destroy the new insulin producing cells introduced either by regenerating, reprogramming or replacement. Several approaches are currently under evaluation for restoration of beta cell mass. The prototype strategy for Replacement is pancreatic islet transplantation, which is now an approved procedure in several countries. Reprogramming from non insulin-producing cells or Regeneration strategies could represent an appealing alternative to overcome shortage of deceased donor organs for transplantation. The selection of the most appropriate source for insulin producing cells is still not defined and the selected alternatives between replacement, reprogramming and regeneration strategies will be further developed in pre-clinical model systems and pilot clinical trials, while carefully assessing safety, efficacy and cost-effectiveness, as well as the challenges imposed by scaling up the selected technologies to meet the demand of the millions of affected patients who could benefit from these strategies.

Anti-inflammatory properties of exenatide in human pancreatic islets.

Exenatide is an analog of the incretin hormone glucagon-like peptide (GLP-1) that is used for the treatment of T2D for their metabolic effects. In addition to its insulinotropic effects, exenatide increases functional islet mass and improves their survival. Improved outcomes have been reported in recent clinical islet transplantation trials for the treatment of type 1 diabetes. The purpose of this study was to investigate whether exenatide has anti-inflammatory properties in human islets. Exenatide treatment improved islet function, significantly reduced content of inflammation-related molecules (tissue factor, IFN-γ, IL-17, IL-1ß, and IL-2) and caspase 3 activation, whereas increased phosphorylation of ERK1/2, STAT3, and Akt in vitro. Immunostaining showed expression of GLP-1R in ß-cells but not in α-cells. IL-1ß colocalized with GLP-1R in ß-cells. Induction of serine proteinase inhibitor 9 (PI-9) was detected after exposure of human islets to exenatide in vitro and after transplantation into immunodeficient mice. GLP-1 induced PI-9 expression in vitro but to a lower extent than exenatide. This effect was partially blocked by the antagonist exendin-9 in vitro. As assessed by immunostaining PI-9 is mostly expressed in ß-cells but not in α-cells. In conclusion, we describe anti-inflammatory and cytoprotective properties of exenatide in human islets. Exenatide-mediated PI-9 expression, the only known granzyme B inhibitor, unveils potential immunoregulatory properties.

Effects of pancreas cold ischemia on islet function and quality.

We used a rat model of pancreas cold preservation to assess its effects on islets. Glands were surgically retrieved and stored in University of Wisconsin (UW) solution for 3 hours (Short) or 18 hours (Long) cold ischemia time (CIT). Islet yield was significantly lower in the Long-CIT than the Short-CIT group, as well as islet recovery after overnight culture (P < .01). Islet cell viability after isolation was significantly reduced in the Long-CIT group (P < .05). Reversal of diabetes following transplantation of suboptimal islet grafts occurred earlier in the Short-CIT group than the Long-CIT. All animals in the Short-CIT group and 80% in the Long-CIT group achieved euglycemia. Freshly isolated islets showed a significant increase of JNK and p38 (P < .05) phosphorylation in Long-CIT compared with Short-CIT. Histopathological assessment of the pancreas showed a significantly higher injury score. Proteomic analysis of pancreatic tissue led to identification of 5 proteins consistently differentially expressed between Short-CIT and Long-CIT. Better understanding of the molecular pathways involved in this phenomenon will be of assistance in defining targeted interventions to improve organ use in the clinical arena.

Ischemic preconditioning improves islet recovery after pancreas cold preservation.

Increasing evidence supports the beneficial effects of ischemic preconditioning (IPC) of organs on subsequent ischemia. The aim of this study was to assess the effects of IPC of the pancreas on islet cell recovery after cold preservation using a rat model. The pancreas was deprived of perfusion (celiac artery and superior mesenteric artery occlusion) for 10 minutes followed by 10 minutes of reperfusion. Islet isolation was performed after 18 hours of cold ischemia. Glands undergoing IPC yielded significantly greater numbers of islets than controls. Following overnight culture, a significantly greater proportion of islets was recovered from IPC-treated pancreata. Microarray genomic analysis of pancreatic tissue revealed a significant differential expression of approximately 600 unique mRNA strands within IPC pancreata compared to only <100 unique mRNA strands within non-IPC pancreata (>2-fold change; P < .05). Proteomic analysis revealed significant differential expression of at least 5 proteins >1.5-fold change; P < .05) within the IPC vs control group. Our data indicated that IPC of the pancreas prior to cold preservation was associated with improved islet cell recovery after cold ischemia. IPC of the pancreas may represent a viable therapeutic intervention to increase islet transplantation success from a single donor and to maximize organ utilization.

c-Jun N-terminal kinase 1 is deleterious to the function and survival of murine pancreatic islets.

AIMS/HYPOTHESIS: Inhibition of c-jun N-terminal kinase (JNK) favours pancreatic islet function and survival. Since two JNK isoforms are present in the pancreas (JNK1 and JNK2), we addressed their specific roles in experimental islet transplantation. METHODS: C57BL/6J (wild-type [WT]), Jnk1 (also known as Mapk8)(-/-) and Jnk2 (also known as Mapk9)(-/-) mice were used as donor/recipients in a syngeneic islet transplantation model. Islet cell composition, function, viability, production of cytokines and of vascular endothelial growth factor (VEGF) were also studied in vitro. RESULTS: Jnk1 ( -/- ) islets secreted more insulin in response to glucose and were more resistant to cytokine-induced cell death compared with WT and Jnk2 (-/-) islets (p < 0.01). Cytokines reduced VEGF production in WT and Jnk2 (-/-) but not Jnk1 ( -/- ) islets; VEGF blockade restored Jnk1 ( -/- ) islet susceptibility to cytokine-induced cell death. Transplantation of Jnk1 ( -/- ) or WT islets into WT recipients made diabetic had similar outcomes. However, Jnk1 ( -/- ) recipients of WT islets had shorter time to diabetes reversal (17 vs 55 days in WT, p = 0.033), while none of the Jnk2 (-/-) recipients had diabetes reversal (0% vs 71% in WT, p = 0.0003). Co-culture of WT islets with macrophages from each strain revealed a discordant cytokine production. CONCLUSIONS/INTERPRETATION: We have shown a deleterious effect of JNK2 deficiency on islet graft outcome, most likely related to JNK1 activation, suggesting that specific JNK1 blockade may be superior to general JNK inhibition, particularly when administered to transplant recipients.

CD40 activation in human pancreatic islets and ductal cells.

AIMS/HYPOTHESIS: CD40 expression on non-haematopoietic cells is linked to inflammation. We previously reported that CD40 is expressed on isolated human and non-human primate islets and its activation results in secretion of IL-8, macrophage inflammatory protein 1-beta (MIP-1beta) and monocyte chemoattractant protein-1 (MCP-1) through nuclear factor-kappaB and extracellularly regulated kinases 1/2 pathways. The objective of this study was to identify the pattern of gene expression, and to study viability and functionality affected by CD40-CD40 ligand (CD40L) interaction in human islets. Furthermore, we have studied the CD40-mediated cytokine/chemokine profile in pancreatic ductal cells, as they are always present in human islet transplant preparations and express CD40 constitutively. METHODS: CD40-CD40L gene expression modulation was studied by microarray on islet cells depleted of ductal cells. Selected genes were validated by quantitative RT-PCR. The cytokine profile in purified ductal cells was evaluated by Luminex technology, based on the use of fluorescent-coated beads, known as microspheres, and capable of multiplex detection of proteins from a single sample. Glucose-stimulated insulin secretion and islet viability were assessed by perifusion and 7-aminoactinomycin D membrane exclusion, respectively. RESULTS: Statistical analysis of microarrays identified 30 genes exhibiting at least a 2.5-fold increase across all replicate arrays. The majority of them were related to oxidative stress/inflammation. Prominently upregulated were chemokine C-X-C motif ligand 1 (CXCL1), CXCL2 and CXCL3 belonging to the CXC family of chemokines related to IL-8. CD40-mediated CXCL1 secretion was confirmed by ELISA. The viability or in vitro function was not affected by CD40 activation. In addition to previously reported IL-8, MIP-1beta and MCP-1, CD40 stimulation in ductal cells produced IL-1beta, IFN-gamma, TNF-alpha, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. CONCLUSIONS/INTERPRETATION: CD40 activation in islets and ductal cells produces cytokines/chemokines with a broad-spectrum range of biological functions.

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation.

AIMS/HYPOTHESIS: Activation of c-jun N-terminal kinase (JNK) has been described in islet isolation and engraftment, making JNK a key target in islet transplantation. The objective of this study was to investigate if JNK inhibition with a cell-permeable TAT peptide inhibitor (L-JNKI) protects functional beta cell mass in human islets and affects AKT and its substrates in islet cells. METHODS: The effect of L-JNKI (10 micromol/l) on islet count, mitochondrial membrane potential, glucose-stimulated insulin release and phosphorylation of both AKT and its substrates, as well as on reversal of diabetes in immunodeficient diabetic Nu/Nu mice was studied. RESULTS: In vitro, L-JNKI reduced the islet loss in culture and protected from cell death caused by acute cytokine exposure. In vivo, treatment of freshly isolated human islets and diabetic Nu/Nu mice recipients of such islets resulted in improved functional beta cell mass. We showed that L-JNKI activates AKT and downregulates glycogen synthase kinase-3 beta (GSK-3B) in human islets exposed to cytokines, while other AKT substrates were unaffected, suggesting that a specific AKT/GSK-3B regulation by L-JNKI may represent one of its mechanisms of cytoprotection. CONCLUSIONS/INTERPRETATION: In conclusion, we have demonstrated that targeting JNK in human pancreatic islets results in improved functional beta cell mass and in the regulation of AKT/GSK3B activity.

Protection of islets in culture by delivery of oxygen binding neuroglobin via protein transduction.

Islet transplantation has become an accepted method to treat type 1 diabetes. To succeed and achieve normal levels of glucose in transplant recipients, the quality of the transplanted islets is of the utmost importance. Lack of oxygen during organ procurement, islet isolation, and subsequent culture triggers apoptosis or necrosis and loss of islet function, causing the yield and quality to diminish. A promising candidate for cytoprotection against oxygen deprivation is neuroglobin (Ngb). Ngb is a recently described member of globin family and is expressed in neurons, retina, and pancreatic islets. To overexpress this protein in the islets and study its ability to protect them, we utilized protein transduction. Protein transduction is achieved by fusing Ngb to the TAT/PTD transduction domain, a peptide originated from the HIV transcriptional transactivator protein. Our study proved that TAT-Ngb is an efficient fusion protein capable of protecting the human islets in culture from loss of cell mass and function, thus increasing the quality of transplantable islets. If the islets could be cultured for a longer period of time without suffering harmful effects, it would be possible to precondition the recipient and there would be more time to assess their quality and function before transplantation.

A functional CD40 receptor is expressed in pancreatic beta cells.

AIMS/HYPOTHESIS: Despite differences in function and embryonic origin, pancreatic islet cells and neurons express proteins belonging to the tumour necrosis factor receptor superfamily. While neurons express the CD40 receptor, it is unknown whether islet cells also express it. We investigated CD40 expression in human and mouse pancreatic islets as well as in NIT-1 insulinoma cells. METHODS: CD40 expression was studied by reverse transcriptase polymerase chain reaction, flow cytometry, immunohistochemistry and western blot. Responses mediated by CD40 were assessed by a luciferase gene reporter assay following stimulation with a CD40 agonist antibody. RESULTS: We found that CD40 is expressed in mouse and human pancreatic islet cells. CD40 is expressed by beta cells, and its expression is upregulated by proinflammatory cytokines (IL-1beta, IFN-gamma and TNF-alpha). CD40 signalling in NIT-1 insulinoma cells activates nuclear factor kappa-B, demonstrating that CD40 is functional. CONCLUSIONS/INTERPRETATION: We present evidence that, in addition to immune cell types, mouse and human pancreatic beta cells express CD40. Its expression is upregulated by proinflammatory stimuli, and signalling through this receptor activates NF-kappaB. We suggest that the effects of inflammatory stimuli that affect beta cell function and survival may be also mediated by signalling through the CD40 receptor. Thus, CD40 may have a role in processes associated with islet autoimmunity and transplantation.

Heme oxygenase-1 induction in islet cells results in protection from apoptosis and improved in vivo function after transplantation.

Transplantation of islets of Langerhans represents a viable therapeutic approach for the treatment of type 1 diabetes. Unfortunately, transplanted islets are susceptible to allogeneic recognition and rejection, recurrence of autoimmunity, and destruction by local inflammation at the site of implantation. The last of these phenomena might not only result in functional impairment and death of islet cells but could also contribute to amplifying the subsequent specific immune response. Induction of islet cell protection against inflammation could therefore be postulated to be a powerful means to improve overall graft fate. Heme oxygenase-1 (HO-1) has been described as an inducible protein capable of cytoprotection via radical scavenging and apoptosis prevention. The purpose of the present study was to analyze whether HO-1 upregulation in a beta-cell line and in freshly isolated murine islets could result in protection from apoptosis and improve in vivo functional performance. HO-1 upregulation was induced reproducibly with protoporphyrins and was correlated with protection from apoptosis induced in vitro with proinflammatory cytokines or Fas engagement. Furthermore, in vivo HO-1 upregulation resulted in improved islet function in a model of marginal mass islet transplantation in rodents. Strategies aimed at inducing HO-1 upregulation might result in improved success in islet transplantation.

Proteins linked to a protein transduction domain efficiently transduce pancreatic islets.

The resounding success of a new immunosuppressive regimen known as the Edmonton protocol demonstrates that islet cell transplantation is becoming a therapeutic reality for diabetes. However, under the Edmonton protocol, a single donor does not provide enough islets to attain the insulin independence of a transplant recipient. This limitation is mainly caused by islet apoptosis triggered during isolation. In this study, we describe a highly efficient system of transiently transferring anti-apoptotic proteins into pancreatic islets, thus opening an exciting new therapeutic opportunity to improve the viability of transplantable islets. We fused beta-galactosidase to the 11-amino acid residues that constitute the protein transduction domain (PTD) of the HIV/TAT protein and transduced pancreatic islets ex vivo with this fusion protein in a dose-dependent manner with >80% efficiency. We observed that transduction of the anti-apoptotic proteins Bcl-X(L) and PEA-15 fused to TAT/PTD prevented apoptosis induced by tumor necrosis factor-alpha in a pancreatic beta-cell line, indicating that TAT/PTD anti-apoptotic proteins retained their biological activity. Finally, we demonstrated that TAT-fusion proteins did not affect the insulin secretion capability of islets, as determined by glucose static incubation and by reversion of hyperglycemia in diabetic immunodeficient mice.

Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A.

The cAMP binding domain of the regulatory subunit (R) of Mucor rouxii protein kinase A was cloned. The deduced amino acid sequence was highly homologous in sequence and in size to the corresponding region in fungal and higher eukaryotic regulatory subunits (47-54%), but particularly homologous (62%) to Blastocladiella emersonii, a fungus classified in a different phylum. Amino acids reported to be important for interaction with cAMP, for cooperativity between the two cAMP binding domains, in the general folding of the domain, and for interaction with the catalytic subunit were conserved in all the fungal sequences. Based on either sequence or functional behavior, the M. rouxii R subunit cannot be classified as being more similar to RI or RII of mammalian systems. The M. rouxii protein sequence was modeled using as template the coordinates of the crystallized bovine regulatory subunit type Ialpha. The quality of the model is good. The two backbones could be perfectly overlapped, except for two loop regions of high divergence. The alpha helix C of domain A, proposed to have a strong interaction with the catalytic subunit, contains a leucine replacing a basic residue (arginine or lysine) commonly found in RI or RII. The domains A and B of the M. rouxii regulatory subunit were overexpressed as fusion proteins with GST. GST domain B protein was inactive. GST domain A was active; the kinetic parameters of affinity toward cAMP analogs, site selectivity, and dissociation kinetics of bound cAMP were analogous to the properties of the domain in the whole regulatory subunit.

Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR.

Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized. Therefore, we have chosen reverse transcriptase real-time PCR to quantitate CD95 (Fas) transcripts to test the cleavage efficiency of anti-Fas ribozymes in the mouse insulinoma cell line beta TC-3. Based on the melting-curve analysis of the amplified products, we determined the temperature at which to collect the fluorescent data used for quantification. After constructing a standard curve by plotting the log of the standards' copy number versus their fractional cycle number, the copy numbers of the unknown samples were automatically determined by interpolation of this curve. As we illustrate in this study, it is important, particularly while setting up the technique, to validate the melting-curve profile with standard gel electrophoresis analysis, achieved by matching melting temperature and size of the amplified product. The method is fast and reproducible: Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 hr. The coefficient of variance is 15% in the range of 10(4)-10(6) gene copies. Accordingly, reverse transcriptase (RT) real-time PCR is a technique suitable for screening a large number of ribozymes.

Inhibition of Fas-mediated apoptosis in mouse insulinoma betaTC-3 cells via an anti-Fas ribozyme.

In this study we have designed and constructed an anti-Fas ribozyme and show that it can specifically cleave the Fas mRNA in vitro. Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into betaTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic beta cells, betaTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-gamma. Transfected cells expressed an average of 5000 copies of anti-Fas ribozyme transcript per cell as assessed by reverse transcriptase-real-time PCR. After IL-1/IFN-gamma treatment, betaTC-3 cells transfected with the anti-Fas ribozyme expressed 80% less Fas compared with mock-transfected cells. In addition, the anti-Fas ribozyme also inhibited Fas expression in NIT-1 insulinoma cells and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in betaTC-3 cells expressing the anti-Fas ribozyme correlated with resistance to Fas-mediated apoptosis as determined by the number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the betaTC-3 pancreatic insulinoma cell line and conferring resistance to Fas-mediated apoptosis. We suggest that ribozymes may be potentially useful to engineer resistance to apoptosis in transplantable beta cells, a feature that may significantly improve the survival of islet cell grafts.

Efficient ex vivo inhibition of perforin and Fas ligand expression by chimeric tRNA-hammerhead ribozymes.

Graft-versus-host disease (GVHD) is a feared complication of allogeneic bone marrow transplantation. Research in rodent models has linked perforin and Fas ligand (FasL), two components of independent lytic pathways, with the induction of GVHD. In this study we characterized two hammerhead ribozymes that cleave their target perforin and Fas ligand RNAs with high efficiency in CTLL-2 cells. The perforin and Fas ligand ribozymes were expressed from a tRNA-directed RNA polymerase III promoter that was inserted in an episomal multicopy plasmid derived from papilloma virus. Chimeric anti-perforin and anti-FasL tRNA-ribozymes had sequences engineered in order to have specific secondary structure effects. These sequence modifications allow the formation of a 5' --> 3' stem structure and also place the ribozyme in a flexible bulge region that keeps the ribozyme separated from the tRNA domain. Northern and RT in situ PCR analyses showed high levels of transcription and efficient transportation to the cytoplasm. The expression of perforin and FasL in CTLL-2 cells was significantly reduced as assessed by RNA and protein analyses.

A hammerhead ribozyme that cleaves perforin and fas-ligand RNAs in vitro.

Polyfunctional ribozymes targeting more than one RNA might be ideal for treatment of diseases caused by the expression of more than one mRNA. This is the case of Graft versus Host Disease, which in the mouse model is due to the action of two proteins responsible for different lytic pathways: perforin and fasligand. We have created a bifunctional ribozyme fusing two antiperforin and antifas-ligand hammerhead ribozymes with a stretch of CA dinucleotides. This bifunctional ribozyme is able to recognize and cleave in vitro perforin and fas-ligand mRNA in a specific and selective fashion, with a catalytic efficiency similar to that of its individual components.

Expression of a specific subset of CD44 variant transcripts in NOD pancreatic islets.

Adhesion of lymphocytes to the endothelial venules inside the islets of Langerhans seems to initiate the infiltration of islets in NOD mice. An overexpression of the lymphocyte surface molecule CD44 in infiltrated NOD islets compared with peripheral blood lymphocytes was recently reported. The CD44 protein family includes a variety of molecules generated by alternative RNA splicing from 10 variant exons (v1-v10). By using reverse transcriptase-polymerase chain reaction followed by Southern blotting and hybridization to exon-specific cDNA probes, we investigated the expression of CD44 isoforms in highly purified islets of Langerhans from 4- and 10-week-old NOD mice. At least six CD44 isoforms were strongly overexpressed in NOD islets at 4 and 10 weeks when compared with age-matched BALB/c islets. Controls in different tissues indicate that these variants are specifically increased in the islets from the NOD strain. Islets from the NOD-scid/scid strain also expressed these variant exons. Splenocytes from BALB/c did not express CD44 isoforms, whereas splenocytes from 4-week-old NOD mice did express CD44 variants. Treatment with inflammatory mediators induced new isoforms; however, these transcripts have a different variant exon composition from that found in NOD mice islets. These results suggest that some isoforms are expressed very early in the development of insulitis by a component of the NOD islet itself and underscore a possible role of CD44 in islet infiltration.