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.

The recent discovery that genetically modified α cells can regenerate and convert into β-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-β-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into β-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated β-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in β-like cell counts, suggestive of α-to-β-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated β-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.

Nutrient homeostasis is known to be regulated by pancreatic islet tissue. The function of islet ␤-cells is controlled by a glucose sensor that operates at physiological glucose concentrations and acts in synergy with signals that integrate messages originating from hypothalamic neurons and endocrine cells in gut and pancreas. Evidence exists that the extrapancreatic cells producing and secreting these (neuro)endocrine signals also exhibit a glucose sensor and an ability to integrate nutrient and (neuro)hormonal messages. Similarities in these cellular and molecular pathways provide a basis for a network of coordinated functions between distant cell groups, which is necessary for an appropriate control of nutrient homeostasis. The glucose sensor seems to be a fundamental component of these control mechanisms. Its molecular characterization is most advanced in pancreatic ␤-cells, with important roles for glucokinase and mitochondrial oxidative fluxes in the regulation of ATPsensitive K + channels. Other glucose-sensitive cells in the endocrine pancreas, hypothalamus, and gut were found to share some of these molecular characteristics. We propose that similar metabolic signaling pathways influence the function of pancreatic ␣-cells, hypothalamic neurons, and gastrointestinal endocrine and neural cells.

Glucose homeostasis is controlled by a glucose sensor in pancreatic fl-cells. Studies on rodent fl-cells have suggested a role for GLUT2 and glucokinase in this control function and in mechanisms leading to diabetes. Little direct evidence exists so far to implicate these two proteins in glucose recognition by human (3-cells. The present in vitro study investigates the role of glucose transport and phosphorylation in fl-cell preparations from nondiabetic human pancreata. Human fl-cells differ from rodent f8-cells in glucose transporter gene expression (predominantly GLUT1 instead of GLUT2), explaining their low K. (3 mmol/liter) and low V m^x (3 mmol/min per liter) for 3-0-methyl glucose transport. The 100-fold lower GLUT2 abundance in human versus rat fl-cells is associated with a 10-fold slower uptake of alloxan, explaining their resistance to this rodent diabetogenic agent. Human and rat f-cells exhibit comparable glucokinase expression with similar flux-generating influence on total glucose utilization. These data underline the importance of glucokinase but not of GLUT2 in the glucose sensor of human fl-cells. (J. Clin. Invest 96:2489-2495

Regeneration, or neogenesis, of beta cells in adult pancreas is an important research issue because it could find applications in the restoration of the normal beta-cell mass in diabetic patients. In vitro neogenesis might be a means of generating additional beta cells intended for transplantation, as the number of beta cells that can be isolated from organ donors is very limited. Neogenesis can be induced in vivo in several ways [1] but the identity of the beta-cell precursors is not clear. Several lines of evidence suggest that, in addition to duct cells, exocrine acinar cells could be able to transdifferentiate into beta cells [2]. In several experimental models and pathological conditions, islet neogenesis is accompanied by the transformation of the normal exocrine tissue into ductal complexes, a process called acinoductal metaplasia. This is seen in the model of pancreatic duct ligation [3], transforming growth factor-alpha transgenic mice [4,5] and interferon-gamma transgenic mice [6]. Islet neogenesis has also been observed in associ- Diabetologia (2000) Abstract Aims/hypothesis. In adult pancreatic regeneration models exocrine acini are found to transdifferentiate to duct-like complexes. This has also been associated with the formation of new endocrine islet cells. We aimed to establish an in vitro model in which this transdifferentiation process is characterised and can be modulated. Methods. Purified rat pancreatic acini were cultured in suspension. Differentiation was analysed by immunocytochemistry, electron microscopy, western blotting and RT-PCR.Results. During culture acinar cells directly transdifferentiated without dividing, the cells lost their acinar phenotype and started to express cytokeratins 20 and 7 and fetal liver kinase-1 (Flk-1) receptors for vascular endothelial growth factor. Expression of the acinar pancreatic exocrine transcription factor (PTF-1) remained and the pancreatic duodenal homeoboxcontaining transcription factor (PDX-1) was induced. When transdifferentiation was completed, the cells started to express protein gene product 9.5, a panneuroendocrine marker. By combining these features, the transdifferentiated cells show similar characteristics to precursor cells during active beta-cell neogenesis. We were able to modulate the differentiation state by addition of nicotinamide or sodium butyrate, agents which are known to stimulate endocrine differentiation in other models. Conclusion/interpretation. Here, we present an in vitro system in which the cellular differentiation of putative pancreatic endocrine precursor cells and their PDX-1 expression can be modulated, thereby providing a possible model for the study of beta-cell transdifferentiation. [Diabetologia (2000) Corresponding author: L. Bouwens, VUB-Department of Experimental Pathology, Laarbeeklaan 103, B-1090 Brussels, Belgium Abbreviations: BrdU, Bromodeoxyuridine; CK7, cytokeratin 7; CK20, cytokeratin 20; Flk-1, fetal liver kinase-1; PDX-1, pancreatic duodenal homeodomain containing transcription factor; PGP9.5, ...

Rat pancreatic a-and P-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in P-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in p-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1(7-36) amide (GLP-I), while RNA from islet a-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in P-cells and not in a-cells. Receptor activity, measured as cellular cAMP production after exposing islet P-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/1 GLP-I and 50 pmol/1 GIP but required 1 nmol/1 glucagon. EC 50 values of GLP-I-and GIP-induced cAMP formation were comparable (0.2 nmol/1) and 45-fold lower than the EC g0 of glucagon (9 nmol/1). Maximal stimulation of cAMP production was comparable for the three peptides. In purified a-cells, 1 nmol/1 GLP-I failed to increase cAMP levels, while 10 pmol/1 to 10 nmol/1 GIP exerted similar stimulatory effects as in P-cells. In conclusion , these data show that stimulation of glucagon,

SummaryGenerating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDHhi), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDHlo cells. Interestingly, gene expression profiling revealed that ALDHhi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS+) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDHhi cells formed INS+ cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential.

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