Increased Intracellular Calcium Is Required for Spreading of Rat Islet β-Cells on Extracellular Matrix

Abstract

Rat islet ␤-cells spread in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G). Furthermore, in a mixed population of cells, it has been observed previously that spread cells secrete more insulin acutely in response to glucose, compared with cells that remain rounded. These results suggest bi-directional signaling between the islet ␤-cell and the extracellular matrix. In the present study, the role of increased intracellular free Ca E xtracellular matrix (ECM) surrounds pancreatic islets and forms the basal lamina, which separates endocrine cells from capillaries. Although the molecular components of these basal laminae remain to be fully investigated in islets, there is evidence indicating that some of the molecules generally found in epithelial basal lamina play a role in the growth and differentiation of ␤-cells. Fetal pancreatic epithelia cultured in Matrigel or collagen I gel differentiated into endocrine cells and form islet structures (1); neonatal rat islet cells embedded in collagen I formed well-organized islet-like organoids (2); and adult human ␤-cells or associated islet cells proliferated when attached on 804G matrix produced by a rat bladder carcinoma cell line (3,4). More recently, it has been shown that laminin I specifically promotes differentiation of mouse ␤-cells from precursor cells in vitro. This finding is of physiological relevance because laminin I is expressed in basement membranes of the mouse pancreas from ϳ15 days of intrauterine life (5).The importance of cell-matrix interactions for an optimal regulated insulin secretion is supported by a number of studies on ␤-cells cultured on various crude matrixes or their purified extracts (6 -14). In a recent study, we observed that ␤-cells attached to a matrix produced by a rat bladder carcinoma cell line 804G (hereafter referred to as 804G matrix) secreted more insulin than control cells. We also noticed that on 804G matrix, cells that had spread expressed higher amounts of ␣6␤1 integrins and secreted more insulin than cells that had remained round (15). The importance of cell-matrix interactions for regulated insulin secretion was further demonstrated by the fact that secretagogues, including glucose, increased the number of spread cells (15).The mechanism leading stimulated ␤-cell to spread on 804G matrix is totally unknown. In the present study, we raise the hypothesis that some of the intracellular pathways governing stimulation-secretion coupling of ␤-cells might be shared for coupling stimulation and spreading. Intracellular free Ca 2ϩ concentration [Ca 2ϩ ] i plays a central role in the consensus model for stimulus-secretion coupling, which may be summarized as follows: in response to glucose, ␤-cell metabolism is stimulated, resulting in increased levels of ATP produced by the mitochondria. ATP-sensitive K ϩ (K ATP ) channels then close, thereby triggering membrane depolarization. The subsequent opening of voltage-dependent Ca 2ϩ channels leads to an increase in [Ca 2ϩ ] i , ...

Cadherin Engagement Improves Insulin Secretion of Single Human β-Cells

“…Cell spreading in response to E-cadherin engagement was inhibited with blocking anti-E-cadherin antibodies or calcium deprivation, indicating that, at least for E-cadherin, spreading did not result from nonspecific interactions. b-Cell spreading on laminin-332 has been described in rodents (25,26) and humans (27). We have demonstrated for the first time that b-cells have the ability to spread when attached to cadherins, suggesting that cadherins and integrins share similar outside-in signaling pathways involved in cytoskeleton remodeling and other cell processes related to cell-shape modification.…”

“…In rat b-cells, we previously observed that spreading cells secreted more insulin in response to glucose compared with round cells (25). This effect was attributed to outside-in signaling through b1 integrin, which induces phosphorylation of focal adhesion kinase (FAK), a protein that localizes at focal contacts (26,31). Despite distinct molecular structures, some crossroads exist between integrins and cadherins (32).…”

“…Therefore, the mechanisms involved in cadherin-mediated cell spreading are likely related to glucose metabolic and/or secretory processes. In rodents, glucose-induced spreading of b-cells on laminin-332 requires an increase in intracellular calcium concentration and is not a consequence of secretory processes that follow elevation of Ca 2+ concentration (26). Calcium mobilization could be also involved in cadherin-mediated cell spreading of human b-cells.…”

Cell–Matrix Interactions Improve β-Cell Survival and Insulin Secretion in Three-Dimensional Culture

“…1 Studies of rat b-cells cultured on matrix produced using a rat bladder carcinoma line (804G) focused on specific integrin interactions and their effect on cell survival, spreading, and insulin secretion. 3,16 The integrin a 6 b 1 interacted with laminin in the 804G-secreted matrix and influenced b-cell function. 3 Similar to studies with cell-derived matrices, culture experiments with purified individual ECM proteins resulted in better islet survival and function.…”

Blockade of β1 Integrin–Laminin-5 Interaction Affects Spreading and Insulin Secretion of Rat β-Cells Attached on Extracellular Matrix

“…We have shown that isolated rat islet ␤-cells cultured on the matrix produced by 804G cells (referred to as 804G matrix) secrete twofold more insulin in response to glucose compared with cells cultured on plastic or poly-L-lysine (22,23). This matrix, deposited by 804G rat bladder carcinoma cells, is rich in laminin-5 (24 -26), a heterotrimer protein consisting of ␣3, ␤3, and ␥2 subunits, and an essential component of several epithelial basement membranes (27,28).…”