Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.

Perturbation of endoplasmic reticulum (ER) homeostasis impairs insulin biosynthesis, beta cell survival, and glucose homeostasis. We show that a murine model of diabetes is associated with the development of ER stress in beta cells and that treatment with the GLP-1R agonist exendin-4 significantly reduced biochemical markers of islet ER stress in vivo. Exendin-4 attenuated translational downregulation of insulin and improved cell survival in purified rat beta cells and in INS-1 cells following induction of ER stress in vitro. GLP-1R agonists significantly potentiated the induction of ATF-4 by ER stress and accelerated recovery from ER stress-mediated translational repression in INS-1 beta cells in a PKA-dependent manner. The effects of exendin-4 on the induction of ATF-4 were mediated via enhancement of ER stress-stimulated ATF-4 translation. Moreover, exendin-4 reduced ER stress-associated beta cell death in a PKA-dependent manner. These findings demonstrate that GLP-1R signaling directly modulates the ER stress response leading to promotion of beta cell adaptation and survival.

Islet grafts can induce insulin independence in type 1 diabetic patients, but their function is variable with only 10% insulin indepence after 5 years. We investigated whether cultured grafts with defined ␤ cell number help standardize metabolic outcome.

The ability of ( cells to endure assaults may be relevant in the development of insulin-dependent diabetes mellitus. This study exas the susceptibility of hu pancreatic islets to agents that are cytotoxic for rodent ( cells-i.e., sodium nitrprusside (NP, a The clinical outbreak of insulin-dependent diabetes mellitus (IDDM) is probably preceded by a long prediabetic period (1), characterized by a delicate balance between autoimmune-induced (3-cell damage (1, 2) and (3-cell repair (3). Among the potential mediators of (3-cell damage, cytokines and cytokine-induced nitric oxide (NO) production have received special attention (4-8). It has been suggested that these agents damage rat islets in vitro (4-8), but their effects on cultured human islets are less pronounced, despite production of similar amounts of NO (9). These observations raise the question whether species differences exist in the susceptibility of pancreatic (3 cells to injury. To assess this possibility, we exposed cultured human, rat, or mouse pancreatic islets to sodium nitroprusside (NP; an NO donor) (10), streptozotocin (SZ; an alkylating agent) (11), or alloxan (a generator of oxygen free radicals) (12) and evaluated the effects of these toxins on islet function. The in vitro cytotoxicity of alloxan and SZ was measured on purified human or rat ,B cells, by using a cell viability test after 1-4 days of subsequent culture (13). Finally, the in vivo toxicity of alloxan was compared in nude mice that had received a graft of either human or mouse pancreatic islets under the kidney capsule.MATERIALS AND METHODS Islet Isolaton. Ten human pancreata were excised from organ donors and transported to the Central Unit ofthe (-Cell Transplant, Brussels, and the islets were isolated as described (14). The age of the donors (mean ± SEM) was 26 + 3 years (range, 9-42 years). Aliquots of the islet-enriched fraction were examined by electron microscopy (n = 10), which indicated 5 ± 1% dead cells and 1 ± 1% acinar cells in the preparations. The prevalence of insulin-and glucagonpositive cells was evaluated by light microscopical examination of immunocytochemically stained islets (15), revealing 57 ± 4% insulin-positive cells and 10 ± 1% glucagon-positive cells. The islet insulin content was 1.60 ± 0.27 ng of insulin per ng of DNA. Human or Wistar rat pancreatic ( cells were purified in Brussels by autofluorescence-activated cell sorting in a FACS-IV (Becton Dickinson) (15,16) and tested locally in the cytotoxicity studies. The isolation of adult rat (Sprague-Dawley) and mouse (NMRI) islets in Uppsala has been described (17). NMRI islets cultured as in the present series of experiments present a prevalence of 77% insulinpositive cells (18).In Vitro Tests. After isolation, human islets were cultured in Brussels in Ham's F-10 medium containing 6

Aims/hypothesis Alginate-encapsulated human islet cell grafts have not been able to correct diabetes in humans, whereas free grafts have. This study examined in immunodeficient mice whether alginate-encapsulated graft function was inferior to that of free grafts of the same size and composition. Methods Cultured human islet cells were equally distributed over free and alginate-encapsulated grafts before implantation in, respectively, the kidney capsule and the peritoneal cavity of non-obese diabetic mice with severe combined immunodeficiency and alloxan-induced diabetes. Implants were followed for in vivo function and retrieved for analysis of cellular composition (all) and insulin secretory responsiveness (capsules). Results Free implants with low beta cell purity (19±1%) were non-functional and underwent 90% beta cell loss. At medium purity (50±1%), they were functional at post-transplant week 1, evolving to normoglycaemia (4/8) or to C-peptide negativity (4/8) depending on the degree of beta cell-specific losses. Encapsulated implants immediately and sustainably corrected diabetes, irrespective of beta cell purity (16/16). Most capsules were retrievable as single units, enriched in endocrine cells that exhibited rapid secretory responses to glucose and glucagon. Single capsules with similar properties were also retrieved from a type 1 diabetic recipient at post-transplant month 3. However, the vast majority were clustered and contained debris, explaining the poor rise in plasma C-peptide. Conclusions/interpretation In immunodeficient mice, i.p. implanted alginate-encapsulated human islet cells exhibited a better outcome than free implants under the kidney capsule. They did not show primary non-function at low beta cell purity and avoided beta cell-specific losses by rapidly establishing normoglycaemia. Retrieved capsules presented secretory responses to glucose, which was also observed in a type 1 diabetic recipient.

No abstract

Previous studies on rat beta cells in vitro have suggested that insulin release is synergistically regulated by signalling molecules derived from glucose metabolism on the one hand and adenylate cyclase stimulation by glucagon or related peptides on the other [1±3]. In rodent beta cells, regulation of the cAMPdependent signalling pathway has been shown [3±7] to depend on expression of specific receptors for glucagon-like peptide-1-(7±36) amide (GLP-1), glucosedependent insulinotropic polypeptide (GIP) and glucagon. The gastrointestinal hormones GLP-1 and GIP effectively increase glucose-induced insulin release in vitro [8±10]; their pivotal role as incretin hormones has been recently illustrated in two mouse models with homozygous null mutations in the respective genes [11,12]. It is not clear whether similar mechanisms operate in human beta cells. More than three decades ago insulin release in humans was shown to be dually stimulated by glucose and glucagon [13,14], the glucagon effect appearing independent of enhanced glucose mobilisation from the liver ]-glucagon-amide (n = 8; p < 0.05), indicating participation of endogenously released glucagon in the process of glucose-induced insulin release. The glucagon-receptor antagonist also suppressed the potentiation of glucose-induced insulin release by addition of 10 nmol/l glucagon. Conclusion/interpretation. These data suggest that human beta cells express functional glucagon receptors which can, similar to incretin hormone receptors, generate synergistic signals for glucose-induced insulin secretion.

Islet transplantation can restore insulin production in insulin-dependent diabetic (IDDM) patients in whom this capacity had been lost for many years [1±7]. This observation has been made in kidney and liver recipients, where advantage is taken of the need for a continuous immune suppression. Survival of the grafts is variable, but generally less than 1 year. A prolonged beta-cell function with a state of insulin-independence after one year was achieved in 7 % of the cases recorded by the Islet Transplant Reg- Diabetologia (1998) Summary Islet allografts in insulin-dependent diabetic (IDDM) patients exhibit variable survival lengths and low rates of insulin-independence despite treatment with anti-T-cell antibodies and maintenance immunosuppression. Use of poorly characterized freshly isolated preparations makes it difficult to determine whether failures are caused by variations in donor tissue. This study assesses survival of standardized beta-cell allografts in C-peptide negative IDDM patients on maintenance immunosuppression following kidney transplantation and without receiving anti-T-cell antibodies or additional immunosuppression. Human islets were isolated from pancreatic segments after maximal 20 h cold-preservation. During culture, preparations were selected according to quality control tests and combined with grafts with standardized cell composition (³ 50 % beta cells), viability ( ³ 90 % ), total beta-cell number (1 to 2 × 10 6 /kg body weight) and insulin-producing capacity (2 to 4 nmol × graft ±1 × h ±1 ). Grafts were injected in a liver segment through the repermeabilized umbilical vein. After 2 weeks C-peptide positivity, four out of seven recipients became C-peptide negative; two of them were initially GAD 65 -antibody positive and exhibited a rise in titre during graft destruction. The other three patients remained C-peptide positive for more than 1 year, two of them becoming insulin-independent with near-normal fasting glycaemia and HbA 1 c ; they remained GAD 65 -and islet cell antibody negative. The three patients with surviving grafts presented a history of anti-thymocyte globulin therapy at kidney transplantation. Long-term surviving grafts increased C-peptide release following intravenous glucagon or oral glucose but not following intravenous glucose. Thus, cultured human beta-cells can survive for more than 1 year in IDDM patients on maintenance anti-rejection therapy for a prior kidney graft and without the need for an increased immunosuppression at the time of implantation. The use of functionally standardized beta-cell grafts helps to identify recipient and graft factors which influence their survival and metabolic effects. Insulin-independence can be achieved by injection of 1.5 million beta-cells per kg body weight in a liver segment. These beta-cell implants respond well to adenylcyclase activators but poorly to glucose. [Diabetologia (1998) 41: 452±459]