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Pancreatic beta cells function within an organ system; cell to cell coupling and interactions with other beta cells, other types of cells and with proteins of the basement membrane are vital to their integrated response to nutrient stimuli. This was indicated by the calcium response to glucose stimuli being greater when cells were cultured as three-dimensional islet-like structures than when cultured as a monolayer (Hauge-Evans, et al., 1999). Other studies, such as that of Hopcroft et al. (1985), indicated that basal insulin secretion could be increased by re-aggregation of dispersed islets. Signalling between beta cells is therefore necessary for maintenance of normal beta cell function and insulin secretion. A more recent study by Konstantinova et al. (2007) showed that when the cell density of the insulinoma cell line MIN6 was increased there was a decrease in the basal insulin secretion and an increase in GSIS. This again indicates the importance of cell to cell communication to beta cells.

Gap junctions are channels between cells composed of connexins which enable the transfer of cytoplasmic ions and metabolites. They have been proposed to be involved in the coordination and synchronisation of cellular responses in beta cells (Jain and Lammert, 2009). Vozzi et al. (1995) showed a causal link between

33 loss of gap junctional communication and impairment of insulin secretion. It was demonstrated that separation of beta cells resulted in a decrease in both insulin biosynthesis and secretion, proposed to be due to connexin43 (Cx43) since insulin secreting cell lines with secretory defects often lack Cx43. However, this conclusion has been more recently disputed. Connexin36 (Cx36) has been shown to form gap junctional complexes in beta cells whilst Cx43 has not been found in either the insulin secreting cell line MIN6 or in primary beta cells (Cigliola, et al., 2013; Jain and Lammert, 2009; Serre-Beinier, et al., 2000). It is therefore considered that connexins have a vital role in communication between beta cells and that Cx36 underlies this communication.

The role of Epithelial-cadherin (E-cadherin) in GSIS was indicated by studies showing that down regulation of E-cadherin in MIN6 cells decreased GSIS to similar levels as found in unattached cells. This suggests that E-cadherin may be necessary for synchronisation of glucose stimulated calcium oscillations (Rogers, et al., 2007). Silencing cadherins in MIN6 cells using small interfering RNA also decreased GSIS, whilst exposure to an anti-cadherin antibody blocked the elevation of intracellular calcium in response to glucose stimulation (Jaques, et al., 2008; Yamagata, et al., 2002).

Interactions of beta cells with ECM proteins are also necessary for optimal beta cell function (Weber, et al., 2008). Interactions with collagen IV and laminin improve MIN6 cell survival and the presence of ECM proteins in the extracellular environment also increased insulin secretion (Weber, et al., 2008). ECM proteins in the basement membrane surrounding beta cells promote cell survival. When cells are detached from their natural ECM environment in the pancreas they undergo apoptosis; this can be prevented in vitro by culturing cells on collagen IV or laminin (Pinkse, et al., 2006). Beta cells interact with basement membrane proteins via integrins. Heterodimers containing the β1-integrin have been suggested to affect insulin transcription and secretion, as well as cell proliferation (Kragl and Lammert, 2010). The expression of ECM proteins such as collagen IV has been suggested to regulate insulin secretion with the specific interaction between the α1β1-integrin heterodimer and collagen IV potentiating insulin secretion (Eberhard, et al., 2010; Kaido, et al., 2004; Kragl and Lammert, 2010). The known components of the basement membrane surrounding pancreatic beta cells and the integrin receptors through which they interact with pancreatic beta cells are given in Table 2.

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Table 2: Matrix proteins and their receptors on pancreatic beta cells

Adapted from Weber et al. (2008).

Recently syndecan-4, a cell-surface proteoglycan, has been shown by immunostaining to be present on insulin-positive but not glucagon-positive cells. It has also been shown to be present in MIN6 cells and it was therefore suggested that beta cells form some of the basement membrane components (Cheng, et al., 2012a). The cytoplasmic domain of syndecan-4 binds to phosphatidylinositol 4,5- bisphosphate and has intracellular effects through activation of protein kinase Cα in a calcium independent manner (Simons and Horowitz, 2001). Syndecans have been described as co-receptors as they frequency work alongside other receptors, particularly integrins in interactions with ECM proteins. Syndecans and integrins collaborate in mediating cell adhesion to ECM proteins and the processes these interactions regulate, including cell migration and cell spreading (Xian, et al., 2010). However, syndecan-4 knockout mice showed no significant impairments in pancreatic function; this may indicate that their role is limited, or alternatively, as suggested by Cheng et al. (2012a) that there is isoform redundancy and other syndecan isoforms are upregulated to compensate. Whilst these proteoglycans have been suggested by Cheng et al. (2012a) to be the missing link to maintaining islet longevity after isolation, further work is required to fully understand their role and the interactions of syndecans with integrins in the basement membrane of the islets of Langerhans.

Protein Localisation Integrin Receptor

Collagen IV Basement membrane α1β1

Laminin Basement membrane α1β1, α3β1, α5β1, α6β1

Collagen I ECM αVβ3, α2β1

Fibrinogen Plasma αVβ3

Fibronectin Plasma, ECM αVβ1, αVβ3, α5β1

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