Summary
Our laboratory pioneered in pancreas tissue slices approach to study the function of insulin-secreting beta cells in mice in 2001 (Speier and Rupnik, 2003; Rupnik 2009). Soon after the tissue slice approach has been extended to assess the perinatal development of the endocrine pancreas (Meneghel-Rozzo et al., 2004; Rozzo et al., 2009), normal and diabetic rats (Rose at al., 2007) as well as to establish the function of insulin-producing stem cells (Blyszczuk et al., 2005). Furthermore, using this approach it became clear that we have to revise our understanding of the ATP sensitivity of ATP-dependent K+ channels (KATP) when studied in more intact environments as fresh tissue slices (Speier et al., 2005). Using slices it has been possible to show the role of cell-to-cell communication through Cx36 gap junctions in the activation and deactivation of beta cells (Speier et al., 2007; Rupnik 2009), the role of KATP channel ablation (Tsiaze et al, in preparation) and the role of protein serotonylation for the secretion of insulin in beta cells (Paulmann et al., 2009). Perinatal tissue slices enabled us to visualize the vast network of pancreas innervation which is typically ignored in most other studies (Meneghel-Rozzo et al., 2004; Rupnik 2009). Other endocrine cells, such as alpha cells, could also be studied in the unperturbed environment (Huang et al., 2011). Important collaborations were done to study the role of Bone morphogenetic protein 3 (Bonner et al., 2011), Munc18-1 and Munc18-2 (Mandić et al., 2011) and assess the level of islet revascularization after transplantation (Nyqvist et al., 2011). The aforementioned significant research achievements led us to several important and novel conclusions. First, beta-cells in pancreatic islets live in complex syncytia which form their normal cellulo-social context. Sufficient cell-to-cell electrical coupling ensures coordinated depolarization pattern. The patched beta cell is merely a biosensor to detect electrical activity of its neighborhood (Speier et al., 2007). Second, the extensive innervation of pancreatic islets interconnecting these structures into neural networks that could be, due to its small size, observed in perinatal pancreas, led us to suggest that insulin release can be both triggered and modulated by rich innervation (Meneghel-Rozzo et al, 2004). Third, the complex structure of the pancreatic islet network (Stožer, Korošak and Rupnik, in preparation) and coordinated action develop about two days after birth during fast proliferation of the endocrine tissue (Rozzo et al, 2009). Fourth, the sensitivity of the KATP channels in beta cells in tissue slices is significantly reduced and allows these channels to be sufficiently open at the physiological level of ATP (3-5 mM) and even without PIP2 or oleyl-CoA as previously suggested. KATP channels open and close only due to changes in cytosolic ATP. ADP levels have no influence on the KATP channels even at concentrations 10 times higher than physiological (Speier et al., 2005). And fifth, in well-known model of type 2 diabetes mellitus, Goto-Kakizaki rat (GK rat), the major lesion does not appear to be the impaired glucose metabolism as has been suggested earlier, but in the use of cytosolic Ca2+ (Rose et al., 2007). Systemic glucose intolerance in GK rats seems to depend on intracellular insensitivity of the secretory machinery to Ca2+. This insensitivity is caused by protein kinase C hyperactivity (Rose et al., 2007). In the subsequent studies it turned out that different patterns of the activity of protein kinases significantly modify the sensitivity of the secretory machinery to Ca2+. Specific activation of protein kinase A increases the sensitivity to Ca2+ (Skelin and Rupnik, 2011; Dolenšek et al., 2011). Incretins, such as GLP-1 and GIP, can activate protein kinase A in pancreatic beta cells and we suggested that this as a major mechanism for these hormones to enhance insulin release from beta cells. On the other hand, specific activation of protein kinase C desensitizes the secretory machinery to Ca2+, but enables more insulin to be released after stronger stimuli (Rose et al., 2007; Dolenšek et al., 2011). Still other kinases, e.g. Cdk5 were found to be involved to act on the Ca2+ sensitivity through their action on Munc18 proteins (Mandić et al., 2011). The phosphorylation pattern of proteins involved in the late steps of the exocytotic activity is an effective and rapid way to modulate the rate of hormone release. Recent results suggest the site of action may be as downstream as the fusion pore protein complex (Skelin et al., 2011). Our laboratory produced significant accomplishments with the use of pancreas tissue slices in addition to other endocrine tissue models, like pituitary gland (Sedej et al., 2004; Sedej et al., 2005; Turner et al.,2005; Dudanova et al., 2006; Sedej et al., 2009) and adrenal medulla chromaffin cell (Dolenšek et al., 2011).