Role In Beta-cell Survival Research Articles

Enhanced structure and function of human pluripotent stem cell-derived beta-cells cultured on extracellular matrix.

The differentiation of human stem cells into insulin secreting beta‐like cells holds great promise to treat diabetes. Current protocols drive stem cells through stages of directed differentiation and maturation and produce cells that secrete insulin in response to glucose. Further refinements are now needed to faithfully phenocopy the responses of normal beta cells. A critical factor in normal beta cell behavior is the islet microenvironment which plays a central role in beta cell survival, proliferation, gene expression and secretion. One important influence on native cell responses is the capillary basement membrane. In adult islets, each beta cell makes a point of contact with basement membrane protein secreted by vascular endothelial cells resulting in structural and functional polarization. Interaction with basement membrane proteins triggers local activation of focal adhesions, cell orientation, and targeting of insulin secretion. This study aims to identifying the role of basement membrane proteins on the structure and function of human embryonic stem cell and induced pluripotent stem cell‐derived beta cells. Here, we show that differentiated human stem cells‐derived spheroids do contain basement membrane proteins as a diffuse web‐like structure. However, the beta‐like cells within the spheroid do not polarize in response to this basement membrane. We demonstrate that 2D culture of the differentiated beta cells on to basement membrane proteins enforces cell polarity and favorably alters glucose dependent insulin secretion.

1067-P: Development of Novel Modulators of the GABAA Receptor for Diabetes Therapy

Accumulating data suggest that the GABAergic system may play a key role in beta-cell survival and regeneration in both type 1 and type 2 diabetes. Therefore, the development of new positive allosteric modulators of the GABAA receptors (GABAA-Rs) provides an interesting strategy for an antidiabetic therapy, although central nervous system side effects mediated by α1-GABAA-Rs are a potential concern. Here we synthesized and functionally analyzed a series of low molecular weight thioacrylamides (ThAcs), designed to both positively modulate GABAA-R signalling and show low blood brain barrier penetration. Beta-cell proliferation was increased by several ThAcs at nanomolar concentrations in a rat beta-cell line in vitro (i.e., ∼82% increased EdU incorporation after 24 hours treatment with ThAc HK-4 at 100 nM). The most effective candidate, HK-4, was further characterized. By using radio ligand competition assays and functional patch clamp experiments we analyzed the interaction between HK-4 and GABAA-Rs. In competition assays, we observed a dose-dependent affinity of HK-4 for GABAA-Rs in rat cortical neuron membranes. In electrophysiological patch clamp assays, we determined the modulating activity of HK-4 to individual human GABAA-Rs using HEK293 cells stably expressing different subunit combinations. HK-4 induced very strong potentiation of the current elicited by the natural ligand GABA on GABAA receptors containing combinations of α2β3γ2 (current ∼300% of GABA alone), α3β3γ2 (∼770%) and on α1β3γ2 (∼580%) subunits, respectively. Minimal blood brain barrier penetration of HK-4 was verified by mass spectrometry-based analyses of brain and plasma samples of rats. Additionally, HK-4 shows no major toxicological effects as determined in a 90-day toxicity study with daily dosing, has a favorable pharmacokinetic profile and demonstrates good oral bioavailability. Our data reveal the potential of HK-4 and other ThAcs for further development into antidiabetic drugs. Disclosure B. Hasse: Stock/Shareholder; Self; Algiax Pharmaceuticals GmbH. B. Belgardt: None. M. Hecht: None. R. Wördenweber: None. A. Piechot: None. M. Roden: Advisory Panel; Self; Servier. Board Member; Self; Poxel SA. Consultant; Self; Eli Lilly and Company, Gilead Sciences, Inc., ProSciento, TARGET PharmaSolutions. Research Support; Self; Boehringer Ingelheim International GmbH, Novartis Pharma K.K., Sanofi US. Speaker’s Bureau; Self; Novo Nordisk A/S. J. Eckel: None. Funding EFRE; NRW; European Union

Role of NumbL:Mig6 Interactions in Beta-Cell Death during Glucolipotoxicity

As the development of type 2 diabetes (T2D) requires a loss of functional beta cell mass, preserving functional beta cell mass is essential for preventing T2D. We have focused our efforts on EGF receptor (EGFR) signaling because it controls cellular proliferation, survival, and repair mechanisms—all features relevant to maintaining beta cell mass—and is dampened during glucolipotoxic conditions (GLT) similar to those in T2D. We have established that Mig6, an endogenous feedback inhibitor of EGFR, is induced in both rodent islets cultured in GLT and human T2D islets. In addition, GLT impairs EGFR signaling in rodent and human islets. Further, Mig6 overexpression promotes apoptosis in both rodent beta cell lines and islets. Because Mig6 is an adapter protein and likely functions as a molecular scaffold, here we aimed to define the molecular mechanisms of Mig6 in GLT by identifying the interacting partners of Mig6. We performed mass spectrometry analysis using low-level, flag-tagged Mig6 expression with immunoprecipitation from Ins1-derived 832/13 cells cultured in normal conditions and GLT. We identified that Mig6 interacted with Numb and NumbL, regulators of Notch signaling, in a context dependent manner. Specifically, Mig6 interacted with Numb and NumbL in normal glucose conditions whereas these interactions were disrupted by GLT. However, roles for Numb and NumbL in beta cell function or death are largely unknown. We observed that Numb and NumbL are expressed in both rodent and human islets, and whereas their expression is not altered in GLT, their localization and aforementioned interactions were. To examine their impact in beta cell death, we used siRNA-mediated suppression of Numb and NumbL in 832/13 cells during control and GLT conditions. Whereas Numb suppression had no effect, NumbL suppression reduced apoptosis in GLT. These results suggest that NumbL plays an important role in beta cell survival. Hence, the differential interactions of Mig6 and NumbL may determine beta cell fate during stress. Disclosure H.D. Basavarajappa: None. J.M. Irimia-Dominguez: None. P.T. Fueger: None.

Selective Ablation of Peptide YY Cells in Adult Mice Reveals Their Role in Beta Cell Survival

In the pancreas, peptide YY (PYY) is expressed by a subpopulation of nonbeta cells in the islets of Langerhans. We investigated the function of these cells in the pancreas of adult mice. We generated mice in which administration of diphtheria toxin (DT) led to specific ablation of PYY-expressing cells. We investigated the effects of loss of PYY cells on glucose homeostasis. Loss of PYY cells in adult mice resulted in severe hyperglycemia, which was associated with significant loss of pancreatic insulin and disruption of islet morphology. In vitro administration of DT to isolated islets significantly reduced numbers of PYY-expressing cells and levels of insulin. Administration of either pancreatic polypeptide (a strong agonist of the receptor Y(4)) or PYY(3-36) (a selective agonist of the receptor Y(2)) did not restore loss of pancreatic insulin following administration of DT. However, a long-acting PYY analogue reduced the loss of insulin, and administration of this analogue reduced the hyperglycemia and insulin loss induced by streptozotocin in mice. PYY appears to regulate beta cell function and survival via the receptor Y(1/2). These findings might be developed to treat and prevent loss of beta cells in patients with diabetes mellitus.

Dysregulation of Dicer1 in Beta Cells Impairs Islet Architecture and Glucose Metabolism

microRNAs (miRNAs) play important roles in pancreas development and in regulation of insulin expression in the adult. Here we show that loss of miRNAs activity in beta-cells during embryonic development results in lower beta-cell mass and in impaired glucose tolerance. Dicer1-null cells initially constitute a significant portion of the total beta-cell population. However, during postnatal development, Dicer1-null cells are depleted. Furthermore, wild-type beta cells are repopulating the islets in complex compensatory dynamics. Because loss of Dicer1 is also associated with changes in the distribution of membranous E-cadherin, we hypothesized that E-cadherin activity may play a role in beta cell survival or islet architecture. However, genetic loss of E-cadherin function does not impair islet architecture, suggesting that miRNAs likely function through other or redundant effectors in the endocrine pancreas.

Expression and function of αβ1 integrins in pancretic beta (INS-1) cells

Integrin-extracellular matrix interactions are important determinants of beta cell behaviours. The β1 integrin is a well-known regulator of beta cell activities; however, little is known of its associated α subunits. In the present study, αβ1 integrin expression was examined in the rat insulinoma cell line (INS-1) to identify their role in beta cell survival and function. Seven α subunits associated with β1 integrin were identified, including α1-6 and αV. Among these heterodimers, α3β1 was most highly expressed. Common ligands for the α3β1 integrin, including fibronectin, laminin, collagen I and collagen IV were tested to identify the most suitable matrix for INS-1 cell proliferation and function. Cells exposed to collagen I and IV demonstrated significant increases in adhesion, spreading, cell viability, proliferation, and FAK phosphorylation when compared to cells cultured on fibronectin, laminin and controls. Integrin-dependent attachment also had a beneficial effect on beta cell function, increasing Pdx-1 and insulin gene and protein expression on collagens I and IV, in parallel with increased basal insulin release and enhanced insulin secretion upon high glucose challenge. Furthermore, functional blockade of α3β1 integrin decreased cell adhesion, spreading and viability on both collagens and reduced Pdx-1 and insulin expression, indicating that its interactions with collagen matrices are important for beta cell survival and function. These results demonstrate that specific αβ1 integrin-ECM interactions are critical regulators of INS-1 beta cell survival and function and will be important in designing optimal conditions for cell-based therapies for diabetes treatment.

Pancreatic transcription factors and their role in the birth, life and survival of the pancreatic β cell

In recent years major progress has been made in understanding the role of transcription factors in the development of the endocrine pancreas in the mouse. Here we describe how a number of these transcription factors play a role in maintaining the differentiated phenotype of the beta cell, and in the mechanisms that allow the beta cell to adapt to changing metabolic demands that occur throughout life. Amongst these factors, Pdx1 plays a critical role in defining the region of the primitive gut that will form the pancreas, Ngn3 expression drives cells towards an endocrine lineage, and a number of additional proteins including Pdx1, in a second wave of expression, Pax4, NeuroD1/beta2, and MafA act as beta cell differentiation factors. In the mature beta cell Pdx1, MafA, beta2, and Nkx2.2 play important roles in regulating expression of insulin and to some extent other genes responsible for maintaining beta cell function. We emphasise here that data from gene expression studies in rodents seldom map on to the known structure of the corresponding human promoters. In the adult the beta cell is particularly susceptible to autoimmune-mediated attack and to the toxic metabolic milieu associated with over-eating, and utilises a number of these transcription factors in its defence. Pdx1 has anti-apoptotic and proliferative activities that help facilitate the maintenance of beta cell mass, while Ngn3 may be involved in the recruitment of progenitor cells, and Pax4 (and possibly HNF1alpha and Hnf4alpha) in the proliferation of beta cells in the adult pancreas. Other transcription factors with a more widespread pattern of expression that play a role in beta cell survival or proliferation include Foxo1, CREB family members, NFAT, FoxM1, Snail and Asc-2.

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel.

ATP-sensitive K+ (KATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of KATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the KATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. KATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the KATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.