Franz Martín

From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus

Abstract Islet transplantation as a potential treatment for diabetes has been investigated extensively over the past 10 years. Such an approach, however, will always be limited mainly because it is difficult to obtain sufficiently large numbers of purified islets from cadaveric donors. One alternative to organ or tissue transplantation is to use a renewable source of cells. Stem cells are clonogenic cells capable of both self-renewal and multilineage differentiation. These cells have the potential to proliferate and differentiate into any type of cell and to be genetically modified in vitro, thus providing cells which can be isolated and used for transplantation. Recent studies have given well-defined differentiation protocols, which can be used to guide stem cells into specific cell lineages as neurons, cardiomyocytes and insulin-secreting cells. Moreover, these derived cells have been useful in different animal models. In this regard, insulin-secreting cells derived from R1 mouse embryonic stem cells restore blood glucose concentrations to normal when they are transplanted into streptozotocin-induced diabetic animals. These results show that diabetes could be among the first applications of stem cell therapy. [Diabetologia (2001) 44: 407–415]

Junctional communication of pancreatic beta cells contributes to the control of insulin secretion and glucose tolerance.

Proper insulin secretion requires the coordinated functioning of the numerous beta cells that form pancreatic islets. This coordination depends on a network of communication mechanisms whereby beta cells interact with extracellular signals and adjacent cells via connexin channels. To assess whether connexin-dependent communication plays a role in vivo, we have developed transgenic mice in which connexin 32 (Cx32), one of the vertebrate connexins found in the pancreas, is expressed in beta cells. We show that the altered beta-cell coupling that results from this expression causes reduced insulin secretion in response to physiologically relevant concentrations of glucose and abnormal tolerance to the sugar. These alterations were observed in spite of normal numbers of islets, increased insulin content, and preserved secretory response to glucose by individual beta cells. Moreover, glucose-stimulated islets showed improved electrical synchronization of these cells and increased cytosolic levels of Ca(2+). The results show that connexins contribute to the control of beta cells in vivo and that their excess is detrimental for insulin secretion.

Induction of Differentiation of Embryonic Stem Cells into Insulin‐Secreting Cells by Fetal Soluble Factors

Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that the developing pancreas releases soluble factors responsible for in vitro endocrine pancreatic differentiation from embryonic stem cells (ESCs). A mouse D3 ESC line was transfected with a human insulin promoter/βgeo/phosphoglycerate kinase–hygromycin‐resistant construct. To direct differentiation, cells were cultured for 7 days to form embryoid bodies and then plated for an additional 7 days. During this 14‐day period, besides eliminating leukemia inhibitory factor, cells were cultured in low serum concentration with the addition of conditioned media from embryonic day–16.5 pancreatic buds. Islet cell differentiation was studied by the following: (a) X‐gal staining after neomycin selection, (b) BrdU (bro‐modeoxyuridine) studies, (c) simple and double immunohistochemistry for insulin, C‐peptide, and glucose transporter 2 (Glut‐2), (d) reverse transcription–polymerase chain reaction for insulin and pancreas duodenum homeobox 1 (PDX‐1), (e) insulin and C‐peptide content and secretion assays, (f) intraperitoneal glucose tolerance test, (g) electrophysiology (patch‐clamp studies in inside‐out configuration), and (h) transplantation of differentiated cells under the kidney capsule of streptozotocin‐diabetic mice. The differentiated ESCs showed the following: changes in the mRNA levels of insulin and PDX‐1; coexpression of insulin, C‐peptide, and Glut‐2; glucose and tolbutamide‐dependent insulin and C‐peptide release; K‐channel activity regulated by ATP; and normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study, we establish a battery of techniques that could be used together to properly characterize islet cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental in engineering β cells from stem cells.

A role for calcium release-activated current (CRAC) in cholinergic modulation of electrical activity in pancreatic beta-cells

S. Bordin and colleagues have proposed that the depolarizing effects of acetylcholine and other muscarinic agonists on pancreatic beta-cells are mediated by a calcium release-activated current (CRAC). We support this hypothesis with additional data, and present a theoretical model which accounts for most known data on muscarinic effects. Additional phenomena, such as the biphasic responses of beta-cells to changes in glucose concentration and the depolarizing effects of the sarco-endoplasmic reticulum calcium ATPase pump poison thapsigargin, are also accounted for by our model. The ability of this single hypothesis, that CRAC is present in beta-cells, to explain so many phenomena motivates a more complete characterization of this current.

Taurine supplementation modulates glucose homeostasis and islet function

Taurine is a conditionally essential amino acid for human that is involved in the control of glucose homeostasis; however, the mechanisms by which the amino acid affects blood glucose levels are unknown. Using an animal model, we have studied these mechanisms. Mice were supplemented with taurine for 30 d. Blood glucose homeostasis was assessed by intraperitoneal glucose tolerance tests (IPGTT). Islet cell function was determined by insulin secretion, cytosolic Ca2+ measurements and glucose metabolism from isolated islets. Islet cell gene expression and translocation was examined via immunohistochemistry and quantitative real-time polymerase chain reaction. Insulin signaling was studied by Western blot. Islets from taurine-supplemented mice had: (i) significantly higher insulin content, (ii) increased insulin secretion at stimulatory glucose concentrations, (iii) significantly displaced the dose-response curve for glucose-induced insulin release to the left, (iv) increased glucose metabolism at 5.6 and 11.1-mmol/L concentrations; (v) slowed cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to stimulatory glucose concentrations; (vi) increased insulin, sulfonylurea receptor-1, glucokinase, Glut-2, proconvertase and pancreas duodenum homeobox-1 (PDX-1) gene expression and (vii) increased PDX-1 expression in the nucleus. Moreover, taurine supplementation significantly increased both basal and insulin stimulated tyrosine phosphorylation of the insulin receptor in skeletal muscle and liver tissues. Finally, taurine supplemented mice showed an improved IPGTT. These results indicate that taurine controls glucose homeostasis by regulating the expression of genes required for glucose-stimulated insulin secretion. In addition, taurine enhances peripheral insulin sensitivity.

Glucose Induces Opposite Intracellular Ca2+ Concentration Oscillatory Patterns in Identified α- and β-Cells Within Intact Human Islets of Langerhans

Homeostasis of blood glucose is mainly regulated by the coordinated secretion of glucagon and insulin from α- and β-cells within the islets of Langerhans. The release of both hormones is Ca2+ dependent. In the current study, we used confocal microscopy and immunocytochemistry to unequivocally characterize the glucose-induced Ca2+ signals in α- and β-cells within intact human islets. Extracellular glucose stimulation induced an opposite response in these two cell types. Although the intracellular Ca2+ concentration ([Ca2+]i) in β-cells remained stable at low glucose concentrations, α-cells exhibited an oscillatory [Ca2+]i response. Conversely, the elevation of extracellular glucose elicited an oscillatory [Ca2+]i pattern in β-cells but inhibited low-glucose–induced [Ca2+]i signals in α-cells. These Ca2+ signals were synchronic among β-cells grouped in clusters within the islet, although they were not coordinated among the whole β-cell population. The response of α-cells was totally asynchronic. Therefore, both the α- and β-cell populations within human islets did not work as a syncitium in response to glucose. A deeper knowledge of α- and β-cell behavior within intact human islets is important to better understand the physiology of the human endocrine pancreas and may be useful to select high-quality islets for transplantation.

Nuclear KATP channels trigger nuclear Ca2+ transients that modulate nuclear function

Glucose, the principal regulator of endocrine pancreas, has several effects on pancreatic beta cells, including the regulation of insulin release, cell proliferation, apoptosis, differentiation, and gene expression. Although the sequence of events linking glycemia with insulin release is well described, the mechanism whereby glucose regulates nuclear function is still largely unknown. Here, we have shown that an ATP-sensitive K+ channel (KATP) with similar properties to that found on the plasma membrane is also present on the nuclear envelope of pancreatic beta cells. In isolated nuclei, blockade of the KATP channel with tolbutamide or diadenosine polyphosphates triggers nuclear Ca2+ transients and induces phosphorylation of the transcription factor cAMP response element binding protein. In whole cells, fluorescence in situ hybridization revealed that these Ca2+ signals may trigger c-myc expression. These results demonstrate a functional KATP channel in nuclei linking glucose metabolism, nuclear Ca2+ signals, and nuclear function.

Transforming growth factor (TGF)β, fibroblast growth factor (FGF) and retinoid signalling pathways promote pancreatic exocrine gene expression in mouse embryonic stem cells

Extracellular signalling cues play a major role in the activation of differentiation programmes. Mouse embryonic stem (ES) cells are pluripotent and can differentiate into a wide variety of specialized cells. Recently, protocols designed to induce endocrine pancreatic differentiation in vitro have been designed but little information is currently available concerning the potential of ES cells to differentiate into acinar pancreatic cells. By using conditioned media of cultured foetal pancreatic rudiments, we demonstrate that ES cells can respond in vitro to signalling pathways involved in exocrine development and differentiation. In particular, modulation of the hedgehog, transforming growth factor beta, retinoid, and fibroblast growth factor pathways in ES cell-derived embryoid bodies (EB) resulted in increased levels of transcripts encoding pancreatic transcription factors and cytodifferentiation markers, as demonstrated by RT-PCR. In EB undergoing spontaneous differentiation, expression of the majority of the acinar genes (i.e. amylase, carboxypeptidase A and elastase) was induced after the expression of endocrine genes, as occurs in vivo during development. These data indicate that ES cells can undergo exocrine pancreatic differentiation with a kinetic pattern of expression reminiscent of pancreas development in vivo and that ES cells can be coaxed to express an acinar phenotype by activation of signalling pathways known to play a role in pancreatic development and differentiation.

Diadenosine Polyphosphates: A Novel Class of Glucose-Induced Intracellular Messengers in the Pancreatic β-Cell

Diadenosine polyphosphates are a group of low-weight compounds that increase after exposure to a wide variety of oxidants and have been suggested to act as “alarmones,” alerting the cell to the onset of metabolic stress. We demonstrate here that glucose at concentrations that induce insulin release produce a 30- to 70-fold increase in the concentration of diadenosine triphosphate (Ap3A) and tetraphosphate (Ap4A) in β-cells. Furthermore, Ap3A and Ap4A, at the concentrations found in glucose-stimulated cells, are effective inhibitors of the ATP-regulated K+ channels when applied to the intracellular side of excised membrane patches from cultured β-cells. We suggest that Ap3A and Ap4A act as second messengers mediating a glucose-induced blockade of the pancreatic β-cell ATP-regulated potassium channel.

Role of syntaxin in mouse pancreatic beta cells

SummaryThe role of syntaxin 1, a protein involved in the docking of synaptic vesicles at presynaptic active zones, has been investigated in pancreatic islet cells. Using two different monoclonal antibodies we have shown that syntaxin 1 is present in the pancreatic islet cell microsomal fraction. Furthermore, functional experiments demonstrate that anti-syntaxin antibodies inhibit Ca2+-dependent insulin secretion in permeabilized islet cells. These data indicate that syntaxin 1 is present in the pancreatic beta cell and it is likely to play a functional role in the exocytosis of secretory granules.