Herein we present a method allowing an accurate analysis of islet cell viability in real time which can be applied to cells and tissues in vitro as well as in vivo. For this purpose we combined the addition of live stains with live confocal microscopy. Several fluorescent dyes such as, wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and Rhod-2, were used to assess cell morphology (WGA), mitochondrial activity (TMRM) or intracellular calcium content (Rhod-2). Microscopy was performed with a microlens-enhanced Nipkow disk-based system which allows live confocal data acquisition. A precise and fast assessment of islet cell viability is achievable using the combination of such a live confocal imaging system with the above mentioned fluorescent dyes. The fast data acquisition and the high precision allowing even single organelle visualization in living cells fulfills the requirements for a fast and efficient monitoring of cell quality under different situations in vitro as well as in vivo. The result is a new method which is fast, accurate and versatile making it an ideal tool to study islet cell biology under different conditions such as after their isolation, transport, transplantation or their culture. Although insights into all these different conditions of islet cell biology are possible, and in spite of “having seen a lot” we still are learning and did not win the battle in the field of islet transplantation. However, considering the amount of other live stains available, this method can be extended and adapted to assess other vital parameters such as the generation of oxygen radicals. Therefore, we are convinced that real time live confocal imaging will be one of the key methods for a better understanding of islet cell biology, especially in the whole context of islet transplantation.

The incretin hormone glucagon-like peptide-1 is encoded by the proglucagon gene (gcg) and produced in both the gut and brainstem. While peripheral GLP-1 stimulates pancreatic beta cell proliferation and insulin secretion, brain GLP-1 controls energy homeostasis with a yet-to-be further defined mechanism. Extensive previous investigations have shown that cAMP signaling is important in the transcription of gcg and the secretion of GLP-1. Recent studies in different disciplines have deepened our understanding on the role of cAMP signaling and its crosstalk with other signaling cascades in endocrine and other cell lineages. Here we summarize recent advances from our own laboratory and elsewhere in the following three areas. A) In the gut, cAMP signaling is able to crosstalk with the Wnt signaling pathway in stimulating the production of the incretin hormones. B) In pancreatic beta cells, cAMP signaling mediates the effect of GLP-1 via cross-talking with a major Wnt signaling effector beta-catenin in stimulating beta cell proliferation. In addition, both protein kinase A (PKA) and Exchange Protein Activated by cAMP (Eapc) are involved in the degradation of TxNIP, a mediator of glucotoxicity. C) The T2D risk gene TCF7L2, which encodes another major effector of the Wnt signaling, is expressed in the brainstem gcg-expressing cells, and its functional knockdown leads to altered glucose homeostasis.

Kaitlyn Kirk,1,2 Ergeng Hao,1,2 Laura Martinson,3 Evert Kroon,3 Pamela Itkin-Ansari*,1,2 The major obstacles to widespread application of islet transplantation for the treatment of diabetes are the scarcity of human islets and the need for chronic immunosuppression. We have studied a durable encapsulation device which is safe in humans, can be transplanted subcutaneously, and is retrievable. We determined that a durable encapsulation device protects murine islets from both allograft rejection and autoimmune disease in the NOD model of Type I diabetes. Moreover, in a preclinical trial it was established that the device is also immunoprotective in primates. In order to address the shortage of islets, we investigated the use of human embryonic stem cells (hESC) derived tissue, a virtually unlimited source of cells. Previously Novocell/ViaCyte reported a method for inducing pancreatic epithelium formation from hESC in vitro. In the present study encapsulated hESC derived pancreatic epithelium was transplanted subcutaneously into mice (n=27). Within 7-9 weeks mice exhibited circulating human insulin (measured as C-peptide). From weeks 7 to 15 both the level of glucose stimulated C-peptide and the stimulation index (maximal/ fasted C-peptide) rose significantly. In vivo bioluminescent imaging (BLI) of luciferase expressing cells revealed that cell mass within the device remained constant during the period of dramatic increases in glucose responsive insulin secretion. Together the data provide evidence of robust cell maturation in the device. The level of circulating human insulin achieved from 20uL of packed cells, was sufficient to influence glucose homeostasis in multiple cohorts of animals treated with the drug alloxan to destroy their endogenous -cells (n=21). In animals exhibiting 4000 pM C-peptide, diabetes reversal was complete for 23 days, the longest period tested. The data establish encapsulation of hESC derived pancreatic epithelium as a promising strategy for providing a widely available, non-immunosuppressive, and minimally invasive transplantation therapy for diabetes. 1 UCSD, Department of Pediatrics, La Jolla, CA 2 Sanford-Burnham Institute for Medical Research, La Jolla, CA 3 ViaCyte Inc., San Diego, CA (previously Novocell) This work is supported by JDRF and CIRM.

Recent molecular-level studies have revealed innovative roles of autophagy in cell survival; still the basic mechanism of autophagy remains unexplored. Further, there exist difficulties in detecting and judging the degree of promotion or suppression through the use of markers, which may not reflect dynamics of such intermediates as autophagosomes and autolysosomes. As an alternative, we present a biophysical approach to the study of autophagy dynamics by means of modeling and numerical simulations of the ‘minimal system’ isolated from its extracellular environment. We examine the behaviors of intracellular concentrations of autophagosomes and autolysosomes and probe how the suppression or promotion of autophagosome formation, autolysosome formation, and intralysosomal hydrolysis steps influence the virtual cellular system. Resulting dynamics of resident protein/organelle, abnormal protein/organelle, ATP, and amino acids reveal the crucial role of autophagy in the regulation of cellular homeostasis and quality control.

Johnson JD, Lim GE, Albrecht T, Mehran AE, Templeman NM, Chu KY, Yang YHC, Szabat M, Alejandro EU, Li J We and others have demonstrated that insulin and other locally released autocrine-paracrine factors (Yang 2011 Diabetologia) have potent effects on pancreatic beta-cell survival and function. Our previous work has demonstrated unexpected roles for multiple proteins, including Raf1, Bad, Foxo1 and Pdx1 (Alejandro 2011 FASEB J; Alejandro 2010 Endocrinology; Johnson 2006 PNAS) in autocrine insulin signalling and shown that insulin is most potent in the picomolar range (Johnson 2008 Cell Cycle). Here, we find using mathematical modeling that the monomeric insulin concentration around beta-cells in the intact islet is likely to be in the picomolar range, given the time required to diffuse from its crystalline state. Using fully functional, fluorescently tagged insulin receptors, TIRFM and super-resolution microscopy, we delineate the route of insulin/insulin receptor complex internalization in beta-cells and implicate Cav1 in this process. We further elucidated autocrine insulin signalling mechanisms, by focusing on the 14-3-3 family of adapter proteins, known to control the activity of Raf1, Bad, Bax, and Foxo. Our comprehensive analysis of this family points to important roles for the14-3-3zeta isoform in the control of beta-cell apoptosis through its interactions with Raf1, Bad and Bax. Finally, by comparing Ins1+/-:Ins2-/- mice that are genetically incapable of hyperinsulinemia to Ins1+/+:Ins2-/- littermate controls, we show that autocrine/paracrine insulin action is required for the increase in beta-cell mass observed after a high fat diet by modulating the rate of apoptosis. Together, these studies demonstrate important roles for insulin signalling in the islet and shed light on novel molecular mechanisms.

AE Sariboyaci, PC Demircan, ZS Unal, G Erman, G Gacar, E Karaoz Recent studies on the immunosuppressive nature of mesenchymal stem cells (MSCs) in human, baboon and murine models showed that MSCs are able to suppress the functions of T-, B- and natural killer- (NK-) cells and the maturation of dendritic cells (DC). In the replacement therapy of type 1 diabetes, pancreatic islet (PI) might be utilized against the autoimmune attacks. We aimed to demonstrate suppressive performance of rat pancreatic islet derived stem cells (rPI-SCs) on concanavalin-A (Con-A)-activated T cells (rat spleens) by co-culturing in mixed lymphocyte reactions (MLR) and transwell systems. Proliferation, apoptosis and pro- & anti-inflammatory cytokines of activated T cells, the expression of regulatory T cells (Treg) markers and also some regulatory factors related to rPI-SC were studied in both transwell and MLR are co-cultures systems. In MLR experiments, activated T cells were cultured with rPI-SC for cell-to-cell interactions, and in Transwell experiments, two chambers were separated by a semi-permeable membrane with a pore size of 0.4 µm. Activated T cells were cultured in the upper chamber of the transwell inserts. Anti-proliferative effect of rPI-SC was determined by WST-1 test and apoptotic effect of rPI-SC was determined by Annexin-V and active caspase labelling in transwell and MLR co-culture systems. Increased levels of anti-inflammatory cytokines secretion, like IL-4, IL-10, IL-13, TGF-β1 by rPI-MSCs and IL-4, IL-10 by T cells were measured by ELISA. We also observed decreased expression levels of CD45, CD62L, IFN-γ, TNF-α and increased expression levels of CD25, IL-4, IL-10 by T cells in MLR and transwell systems by flow cytometry. We demonstrated apoptosis of activated T cells within 24 h using by time-lapse camera photographs and by active caspase labeling. As conclusion, rPI-SCs could exert all of these regulatory effects through the increased levels of paracrine soluble factors and anti-inflammatory cytokines when co-cultured with ConA activated T-cells. In this way, these stem or progenitor cells located in pancreatic islets could also show immuneregulatory effect in vivo. Therefore, the study to understand the pathogenesis of type-1 diabetes and to identify the role of these cells in the development of autoimmune attacks should be performed. *This study was supported by grants from the Scientific and Research Council of Turkey (TUBITAK).

G Duruksu, A Okcu, G Gacar, C Subasi, E Karaoz. One of the promising method for the treatment of type-1 diabetes is the cell replacement therapy, focused on the increase of β-cell numbers. Beside of the scarce resource of islets available for transplantation, the grafts were still under the risk of attacks by immune system. Therefore, to understand the intrinsic protection mechanisms of those cells could improve the clinical efforts. For that purpose, indirect coculture system of pancreatic islets with pancreatic islets-derived stem cells was performed in this study. In this system, pancreatic islets and stem cells obtained from the islets were cultured in the same media, but separated by a porous membrane, preventing cell-to-cell contact. Apoptosis was induced in β-cells by means of streptozotocin. Insulin secretions of treated, untreated and cocultured islets were compared and the cytokine release to media were measured, in which IL-6 and TGF-β1 secretions were found to be up-regulated in coculture systems. Highest cytokine values were obtained in the coculture of stem cells with damaged islets. Response to different glucose concentrations was preserved in β-cells by pancreatic islet derived stem cells during the treatment. The results showed that the pancreatic islet derived stem cells protected pancreatic islets by secreting soluble factors and cytokines to reverse the damage of streptozotocin. IL-6 and TGF-β1 are the key factors in this protective effect.

Assessment of β-cell function, simultaneously with insulin sensitivity under physiological conditions, has been a challenge because of its complex interplay with insulin secreting pancreatic β-cell and glucose consuming organs. In this study, we present a mathematical model for the glucose-insulin regulatory system to provide a theoretical basis for the assessment and prediction of diabetes mellitus. Through extensive numerical simulations and analysis, we are able to observe dynamics of the intracellular Ca2+ concentration, membrane potential, and insulin secretion rate in a pancreatic β-cell, coupled with glucose dynamics at the whole-body level, taking into account the hepatic glucose production and the glucose uptake rate in the brain, muscle, and adipose tissue. In addition, glucose dynamics in the whole-body as well as insulin dynamics in the pancreatic β-cell are predicted in response to perturbations in pancreatic β-cell, liver, and peripheral tissues. Finally, we examine how the dynamics of the pancreatic β-cell as well as of the whole-body is affected by the presence of a constant glucose input, which simulates the glucose infusion in glucose clamps or intravenous glucose tolerance tests.