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Abstract: In a previous review for Advances in Enzyme Research () we outlined the history of our involvement in discovering important roles for inositides in the insulin secreting pancreatic beta cell. In this current appraisal we bring the work up to date and project how we believe this field will continue to develop in the future. Recently, we have seen an important synergism between the growth in our understanding of inositide function and our knowledge of beta cell stimulus-secretion coupling in both physiological and pathophysiological contexts. Important advances have been made in three areas. 1. The classic regulation of cytoplasmic free Ca2+ concentration [Ca2+]i by Inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) and its receptor, 2. A novel role of the inositol pyrophosphates, especially 5-diphosphoinositol pentakisphosphate (5-PP-InsP 5), in exocytosis, and 3. The unique signaling roles of PI3K pathways instituted by the engagement of the insulin receptor in an autocrine, positive feed-back loop. We examine each of these in turn and close with an assessment of the likely future directions the research will take. [Copyright &y& Elsevier]

Abstract: Over the last two decades we have focused on beta cell signal transduction, bringing many new insights, especially in the context of insulin signal transduction, the role of inositol polyphosphates and the regulation of cytoplasmic free Ca2+ concentration. However, there has been a growing awareness that the beta cell, which is mandatory for insulin secretion, has a unique context within the micro-organ of the pancreatic Islet of Langerhans. In this environment the beta cell both mediates and receives paracrine regulation, critical for the control of blood glucose homeostasis. Failure of an appropriate beta cell function leads to the development of diabetes mellitus. In our quest to understand the molecular events maintaining beta cell function we have faced two key challenges. Firstly, whilst there are many similarities between signal transduction in pancreatic islets between the much used rodent models and humans there are some notable differences. Critical distinctions between rodent and primate can be made in the structure of the islet, including the arrangement of the islet cells, the innervation pattern and the microcirculation. This means that important signaling interactions between islets cells, mediated through for example insulin, glucagon, GABA, glutamate and ATP, will have a unique human framework. The second challenge was to be able to take the discoveries we have made using in vitro systems and examine them in an in vivo context. Advances in in vivo imaging achieved by utilizing the anterior chamber of the eye as a transplantation site for pancreatic islets make it possible for non-invasive, longitudinal studies at single cell resolution in real time of islet cell physiology and pathology. Thus it is becoming possible to study the insulin secreting pancreatic beta cell within the framework of the unique micro-organ, the Islet of Langerhans, for the first time in a physiological context, i.e. when being innervated and connected to the blood supply. [Copyright &y& Elsevier]

The inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP 7), is thought to negatively regulate the critical insulin signaling protein Akt/PKB. Knockdown of the IP 7 -generating inositol hexakisphosphate kinase 1 (IP6K1) results in a concomitant increase in signaling through Akt/PKB in most cell types so far examined. Total in vivo knockout of IP6K1 is associated with a phenotype resistant to high-fat diet, due to enhanced Akt/PKB signaling in classic insulin regulated tissues, counteracting insulin resistance. In contrast, we have shown an important positive role for IP6K1 in insulin exocytosis in the pancreatic β-cell. These cells also possess functional insulin receptors and the feedback loop following insulin secretion is a key aspect of their normal function. Thus we examined the effect of silencing IP6K1 on the activation of Akt/PKB in β-cells. Silencing reduced the glucose-stimulated increase in Akt/PKB phosphorylation on T308 and S473. These effects were reproduced with the selective pan-IP6K inhibitor TNP. The likely explanation for IP 7 reduction decreasing rather than increasing Akt/PKB phosphorylation is that IP 7 is responsible for generating the insulin signal, which is the main source of Akt/PKB activation. In agreement, insulin receptor activation was compromised in TNP treated cells. To test whether the mechanism of IP 7 inhibition of Akt/PKB still exists in β-cells, we treated them at basal glucose with an insulin concentration equivalent to that reached during glucose stimulation. TNP potentiated the Akt/PKB phosphorylation of T308 induced by exogenous insulin. Thus, the IP 7 regulation of β-cell Akt/PKB is determined by two opposing forces, direct inhibition of Akt/PKB versus indirect stimulation via secreted insulin. The latter mechanism is dominant, masking the inhibitory effect. Consequently, pharmacological strategies to knock down IP6K activity might not have the same positive output in the β-cell as in other insulin regulated tissues. Unlabelled Image • Silencing IP6K1 inhibits glucose-induced Akt/PKB activation in pancreatic β-cells. • A pan IP6K inhibitor, TNP, also reduces glucose-stimulated Akt/PKB phosphorylation. • IP6K1 inhibition impairs Akt/PKB by curtailing insulin secretion and its autocrine feedback. • There are both positive and negative actions of IP 7 on Akt/PKB. • The dominant positive action of IP 7 on Akt/PKB is due to unique insulin autocrine feedback. [ABSTRACT FROM AUTHOR]

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP 7 , is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP 7 regulation in β-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP 7 . Although the inhibitors have diverse targets, they all perturbed IP 7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP 7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K m for ATP makes IP 7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP 7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP 7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic β-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP 7 ’s role in cellular signal transduction is likely to have been underestimated. [ABSTRACT FROM AUTHOR]

Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM-derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro . Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation. [ABSTRACT FROM AUTHOR]

Defects in pancreatic β-cell function and survival are key components in type 2 diabetes (T2D). An age-dependent deterioration in β-cell function has also been observed, but little is known about the molecular mechanisms behind this phenomenon. Our previous studies indicate that the regulation of cytoplasmic free Ca 2+ concentration ([Ca 2+ ] i ) may be critical and that this is dependent on the proper function of the mitochondria. The [Ca 2+ ] i dynamics of the pancreatic β-cell are driven by an interplay between glucose-induced influx of extracellular Ca 2+ via voltage-dependent Ca 2+ channels and the inositol 1,4,5-trisphosphate (Ins(1,4,5) P 3 )-mediated liberation of Ca 2+ from intracellular stores. Our previous work has indicated a direct relationship between disruption of Ins(1,4,5) P 3 -mediated Ca 2+ regulation and loss of β-cell function, including disturbed [Ca 2+ ] i dynamics and compromised insulin secretion. To investigate these processes in aging we used three mouse models, a premature aging mitochondrial mutator mouse, a mature aging phenotype (C57BL/6) and an aging-resistant phenotype (129). Our data suggest that age-dependent impairment in mitochondrial function leads to modest changes in [Ca 2+ ] i dynamics in mouse β-cells, particularly in the pattern of [Ca 2+ ] i oscillations. These changes are driven by modifications in both PLC/Ins(1,4,5) P 3 -mediated Ca 2+ mobilization from intracellular stores and decreased β-cell Ca 2+ influx over the plasma membrane. Our findings underscore an important concept, namely that even relatively small, time-dependent changes in β-cell signal-transduction result in compromised insulin release and in a diabetic phenotype. [ABSTRACT FROM AUTHOR]

We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR).Wenowhypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YFNephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin- Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfateinduced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function. [ABSTRACT FROM AUTHOR]