Bernat Soria

Widespread synchronous [Ca2+]i oscillations due to bursting electrical activity in single pancreatic islets.

Pancreatic β cells, tightly organized in the islet of Langerhans, secrete insulin in response to glucose in a calcium-dependent manner. The calcium input required for this secretory activity is thought to be provided by an oscillatory electrical activity occurring in the form of “bursts” of calcium action potentials. The previous observation that islet intracellular free Ca2+ levels undergo spontaneous oscillations in the presence of glucose, together with the fact that islet cells are coupled through gap junctions, hinted at a highly effective co-ordination between individual islet cells. Through the use of simultaneous recordings of intracellular calcium and membrane potential it is now reported that the islet calcium waves are synchronized with the β cell bursting electrical activity. This observation suggests that each calcium wave is due to Ca2+ entering the cells during a depolarized phase of electrical activity. Moreover, fura-2 fluorescence image analysis indicates that calcium oscillations occur synchronously across the whole islet tissue. The maximal phase shift between oscillations occurring in different islet cells is estimated as 2 s. This highly co-ordinated oscillatory calcium signalling system may underlie pulsatile insulin secretion and the islet behaviour as a secretory “syncytium”. Since increasing glucose concentration lengthens calcium wave and burst duration without significantly affecting wave amplitude, we further propose that it is the fractional time at an enhanced Ca2+ level, rather than its amplitude, that encodes for the primary response of insulin-secreting cells to fuel secretagogues.

Low Doses of Bisphenol A and Diethylstilbestrol Impair Ca2+ Signals in Pancreatic α-Cells through a Nonclassical Membrane Estrogen Receptor within Intact Islets of Langerhans

Glucagon, secreted from pancreatic α-cells integrated within the islets of Langerhans, is involved in the regulation of glucose metabolism by enhancing the synthesis and mobilization of glucose in the liver. In addition, it has other extrahepatic effects ranging from lipolysis in adipose tissue to the control of satiety in the central nervous system. In this article, we show that the endocrine disruptors bisphenol A (BPA) and diethylstilbestrol (DES), at a concentration of 10−9 M, suppressed low-glucose–induced intracellular calcium ion ([Ca2+]i) oscillations in α-cells, the signal that triggers glucagon secretion. This action has a rapid onset, and it is reproduced by the impermeable molecule estradiol (E2) conjugated to horseradish peroxidase (E-HRP). Competition studies using E-HRP binding in immunocytochemically identified α-cells indicate that 17β-E2, BPA, and DES share a common membrane-binding site whose pharmacologic profile differs from the classical ER. The effects triggered by BPA, DES, and E2 are blocked by the Gαi- and Gαo-protein inhibitor pertussis toxin, by the guanylate cyclase–specific inhibitor 1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one, and by the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester. The effects are reproduced by 8-bromo-guanosine 3′,5′-cyclic monophosphate and suppressed in the presence of the cGMP-dependent protein kinase inhibitor KT-5823. The action of E2, BPA, and DES in pancreatic α-cells may explain some of the effects elicited by endocrine disruptors in the metabolism of glucose and lipid.

Rapid insulinotropic effect of 17β-estradiol via a plasma membrane receptor

Impaired insulin secretion is a hallmark in both type I and type II diabetic individuals. Whereas type I (insulin‐dependent diabetes mellitus) implies β‐cell destruction, type II (non‐insulin dependent diabetes mellitus), responsible for 75% of diabetic syndromes, involves diminished glucose‐dependent secretion of insulin from pancreatic β‐cells. Although a clear demonstration of a direct effect of 17β‐estradiol on the pancreatic β‐cell is lacking, an in vivo insulinotropic effect has been suggested. In this report we describe the effects of 17β‐estradiol in mouse pancreatic β‐cells. 17β‐Estradiol, at physiological concentrations, closes KATP channels, which are also targets for antidiabetic sulfonylureas, in a rapid and reversible manner. Furthermore, in synergy with glucose, 17β‐estradiol depolarizes the plasma membrane, eliciting electrical activity and intracellular calcium signals, which in turn enhance insulin secretion. These effects occur through a receptor located at the plasma membrane, distinct from the classic cytosolic estrogen receptor. Specific competitive binding and localization of 17β‐estradiol receptors at the plasma membrane was demonstrated using confocal reflective microscopy and immunocytochemistry. Gaining deeper knowledge of the effect induced by 17β‐estradiol may be important in order to better understand the hormonal regulation of insulin secretion and for the treatment of NIDDM.— Nadal, A., Rovira, J. M., Laribi, O., Leon‐Quinto, T., Andreu, E., Ripoll, C., Soria, B. Rapid insulinotropic effect of 17b‐estradiol via a plasma membrane receptor. FASEB J. 12, 1341–1348 (1998)

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]

Homologous and heterologous asynchronicity between identified α‐, β‐ and δ‐cells within intact islets of Langerhans in the mouse

1 Using laser scanning confocal microscopy to image [Ca2+]i within intact murine islets of Langerhans, we analysed the [Ca2+]i signals generated by glucose in immunocytochemically identified α‐, β‐ and δ‐cells. 2 Glucagon‐containing α‐cells exhibited [Ca2+]i oscillations in the absence of glucose, which petered out when islets were exposed to high glucose concentrations. 3 Somatostatin‐containing δ‐cells were silent in the absence of glucose but concentrations of glucose as low as 3 mM elicited oscillations. 4 In pancreatic β‐cells, a characteristic oscillatory calcium pattern was evoked when glucose levels were raised from 3 to 11 mM and this was synchronized throughout the β‐cell population. Remarkably, [Ca2+]i oscillations in non‐β‐cells were completely asynchronous, both with respect to each other and to β‐cells. 5 These results demonstrate that the islet of Langerhans behaves as a functional syncytium only in terms of β‐cells, implying a pulsatile secretion of insulin. However, the lack of a co‐ordinated calcium signal in α‐ and δ‐cells implies that each cell acts as an independent functional unit and the concerted activity of these units results in a smoothly graded secretion of glucagon and somatostatin. Understanding the calcium signals underlying glucagon and somatostatin secretion may be of importance in the treatment of non‐insulin‐dependent diabetes mellitus since both glucagon and somatostatin appear to regulate insulin release in a paracrine fashion.

Low doses of the endocrine disruptor bisphenol-A and the native hormone 17beta-estradiol rapidly activate transcription factor CREB.

Endocrine‐disrupting chemicals (EDCs) are hormone‐like agents present in the environment that alter the endocrine system of wildlife and humans. Most EDCs have potencies far below those of the natural hormone 17β‐E2 when acting through the classic estrogen receptors (ERs). Here, we show that the environmental estrogen Bisphenol‐A and the native hormone 17β‐E2 activate the transcription factor, cAMP‐responsive element binding protein (CREB) with the same potency. Phosphorylated CREB (P‐CREB) was increased after only a 5‐minute application of either BPA or 17β‐E2 in a calcium‐dependent manner. The effect was reproduced by the membrane‐impermeable molecule E2 conjugated to horseradish peroxidase (E‐HRP). The increase in PCREB was not modified by the anti‐estrogen ICI 182,780. Therefore, low‐dose of BPA activates the transcription factor CREB via an alternative mechanism, involving a non‐classical membrane estrogen receptor. Because these effects are elicited at concentrations as low as 10–9 M, this observation is of environmental and public health relevance.

Glucose-induced oscillations of intracellular Ca2+ concentration resembling bursting electrical activity in single mouse islets of Langerhans

Intracellular Ca2+ levels were monitored in single, acutely isolated mouse islets of Langerhans by dual emission Indo‐1 fluorometry. High‐frequency (3.1 min−1) [Ca2+]i, oscillations with a brief rising time (1–2 s) and 10 s half‐width (‘fast’ oscillations) were detected in 11 mM glucose. Raising the glucose concentration to 16.7 mM increased the duration of these oscillations, which were otherwise absent in 5.5 mM glucose. [Ca2+]i waves of lower frequency (0.5 min−1) and longer rising time (‘slow’ oscillations) were also recorded. The data indicate that “fast” oscillations are directly related to β‐cell bursting electrical activity, and suggest the existence of extensive networks of electrically coupled cells in the islet.

In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells

Aims/hypothesisWe recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. MethodsMouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice.ResultsThe protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols.Conclusions/interpretationThese methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.

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.

Sirtuin 1 regulation of developmental genes during differentiation of stem cells

The longevity-promoting NAD+–dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.