Pancreatic islets are micro-organs composed of a mixture of endocrine and non-endocrine cells, where the former secrete hormones and peptides necessary for metabolic homeostasis. Through vasculature and innervation the cells within the islets are in communication with the rest of the body, while they interact with each other through juxtacrine, paracrine and autocrine signals, resulting in fine-tuned sensing and response to stimuli. In this context, cellular protrusion in islet cells, such as primary cilia and filopodia, have gained attention as potential signaling hubs. During the last decade, several pieces of evidence have shown how the primary cilium is required for islet vascularization, function and homeostasis. These findings have been possible thanks to the development of ciliary/basal body specific knockout models and technological advances in microscopy, which allow longitudinal monitoring of engrafted islets transplanted in the anterior chamber of the eye in living animals. Using this technique in com-bination with optogenetics, new potential paracrine interactions have been suggested. For example, reshaping and active movement of filopodia-like protrusions of 8-cells were visualized in vivo, suggesting a continuous cell remodeling to increase intercellular contacts. In this review, we discuss these recent discoveries regarding primary cilia and filopodia and their role in islet homeostasis and intercellular islet communication.

Multiple inositol polyphosphate phosphatase (MINPP1) is an enigmatic enzyme that is responsible for the metabolism of inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6 pentakisphosphate (Ins(1,3,4,5,6)P5 in mammalian cells, despite being restricted to the confines of the ER. The reason for this compartmentalization is unclear. In our previous studies in the insulin-secreting HIT cell line, we expressed MINPP1 in the cytosol to artificially reduce the concentration of these higher inositol phosphates. Undocumented at the time, we noted cytosolic MINPP1 expression reduced cell growth. We were struck by the similarities in substrate preference between a number of different enzymes that are able to metabolize both inositol phosphates and lipids, notably IPMK and PTEN. MINPP1 was first characterized as a phosphatase that could remove the 3-phosphate from inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). This molecule shares strong structural homology with the major product of the growth-promoting Phosphatidyl 3-kinase (PI3K), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and PTEN can degrade both this lipid and Ins(1,3,4,5)P4. Because of this similar substrate preference, we postulated that the cytosolic version of MINPP1 (cyt-MINPP1) may not only attack inositol polyphosphates but also PtdIns(3,4,5)P3, a key signal in mitogenesis. Our experiments show that expression of cyt-MINPP1 in HIT cells lowers the concentration of PtdIns(3,4,5)P3. We conclude this reflects a direct effect of MINPP1 upon the lipid because cyt-MINPP1 actively dephosphorylates synthetic, di(C4:0)PtdIns(3,4,5)P3 in vitro. These data illustrate the importance of MINPP1′s confinement to the ER whereby important aspects of inositol phosphate metabolism and inositol lipid signaling can be separately regulated and give one important clarification for MINPP1′s ER seclusion.

Glucose-stimulatedinsulin secretion is the hallmark of the pancreatic β-cell, a critical playerin the regulation of blood glucose concentration. In 1974 Dawson, Freinkel andco-workers made the remarkable observation that an efflux of intracellular inorganicphosphate (Pi) accompanied the events of stimulated insulinsecretion. The mechanism behind this ‘phosphate flush’, its association withinsulin secretion and its regulation have since then remained a mystery. Werecapitulated the phosphate flush in the MIN6m9 β-cell line and pseudoislets.We demonstrated that knockdown of XPR1, a phosphate transporter present inMIN6m9 cells and pancreatic islets, prevented this flush. Concomitantly, XPR1silencing led to intracellular Pi accumulation and a potential impacton Ca2+ signaling. XPR1 knockdown slightly blunted first phaseglucose-stimulated insulin secretion in MIN6m9 cells, but had no significantimpact on pseudoislet secretion. In keeping with other cell types, basal Piefflux was stimulated by inositol pyrophosphates and basal intracellular Piaccumulated following knockdown of inositol hexakisphosphate kinases. However,the glucose-driven phosphate flush occurred despite inositol pyrophosphatedepletion. Finally, whilst it is unlikely that XPR1 directly affectsexocytosis, it may protect Ca2+ signaling. Thus we have revealedXPR1 as the missing mediator of the phosphate flush, shedding light on a45-year-old mystery.

The inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), is thought to negatively regulate the critical insulin signaling protein Akt/PKB. Knockdown of the IP7-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 IP7 reduction decreasing rather than increasing Akt/PKB phosphorylation is that IP7 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 IP7 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 IP7 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.

Diphosphoinositol pentakisphosphate (IP7) is critical for the exocytotic capacity of the pancreatic β-cell, but its regulation by the primary instigator of β-cell exocytosis, glucose, is unknown. The high Km for ATP of the IP7-generating enzymes, the inositol hexakisphosphate kinases (IP6K1 and 2) suggests that these enzymes might serve as metabolic sensors in insulin secreting β-cells and act as translators of disrupted metabolism in diabetes. We investigated this hypothesis and now show that glucose stimulation, which increases the ATP/ADP ratio, leads to an early rise in IP7 concentration in β-cells. RNAi mediated knock down of the IP6K1 isoform inhibits both glucose-mediated increase in IP7 and first phase insulin secretion, demonstrating that IP6K1 integrates glucose metabolism and insulin exocytosis. In diabetic mouse islets the deranged ATP/ADP levels under both basal and glucose-stimulated conditions are mirrored in both disrupted IP7 generation and insulin release. Thus the unique metabolic sensing properties of IP6K1 guarantees appropriate concentrations of IP7 and thereby both correct basal insulin secretion and intact first phase insulin release. In addition, our data suggest that a specific cell signaling defect, namely, inappropriate IP7 generation may be an essential convergence point integrating multiple metabolic defects into the commonly observed phenotype in diabetes.

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, IP7, is at a high level in pancreatic β-cells and is important for insulin exocytosis. To better understand IP7 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 IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high Km for ATP makes IP7 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 IP7 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 IP7 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, IP7's role in cellular signal transduction is likely to have been underestimated.

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 Ca2+ concentration ([Ca2+]i) may be critical and that this is dependent on the proper function of the mitochondria. The [Ca2+]i dynamics of the pancreatic β-cell are driven by an interplay between glucose-induced influx of extracellular Ca2+ via voltage-dependent Ca2+ channels and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-mediated liberation of Ca2+ from intracellular stores. Our previous work has indicated a direct relationship between disruption of Ins(1,4,5)P3-mediated Ca2+ regulation and loss of β-cell function, including disturbed [Ca2+]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 [Ca2+]i dynamics in mouse β-cells, particularly in the pattern of [Ca2+]i oscillations. These changes are driven by modifications in both PLC/Ins(1,4,5)P3-mediated Ca2+ mobilization from intracellular stores and decreased β-cell Ca2+ 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.

Studies of inositol polyphosphates in the pancreatic β-cell have led to an exciting synergism between new discoveries regarding their cellular roles and new insights into β-cell function. Because the loss or malfunction of the β-cell is central to diabetes, these studies open the possibility of new pharmacological interventions in a disease that has reached epidemic proportions worldwide. Using the β-cell as our prime but not exclusive example, we examine the inositol polyphosphates in three main groups: 1) inositol 1,4,5-trisphosphate and its influence on Ca(2+) signaling, specifically in a cell in which cytoplasmic-free Ca(2+) concentration is principally increased by plasma membrane standing voltage-gated Ca(2+) channels; 2) higher inositol polyphosphates including a novel second messenger inositol 3,4,5,6-tetrakisphosphate and a regulatory role for inositol hexakisphosphate in β-cell Ca(2+) homeostasis and exo- and endocytosis; and 3) inositol pyrophosphates and their role in β-cell exocytosis, together with the exciting possibility of being novel targets for therapy in diabetes. We conclude with some of the new perspectives that are likely to become apparent in the next few years.

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.

In a previous review for Advances in Enzyme Research (Berggren and Barker, 2008) 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)P3) and its receptor, 2. A novel role of the inositol pyrophosphates, especially 5-diphosphoinositol pentakisphosphate (5-PP-InsP5), 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.

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P(3)], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P(3) is considered to be important to complex Fe(3+) in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P(4)], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe(3+), evident from excimer fluorescence induced by pi-pi stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca(2+) cations exceeding a 1 : 1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe(3+) have been determined. The complexes formed between Fe(3+) and 4,6-bispyrenoyl Ins(1,2,3,5)P(4) display similar stability to those formed with Ins(1,2,3)P(3), indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P(4), which closely resemble those obtained for Ins(1,2,3)P(3). The data presented confirms that Fe(3+) binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif.

Phosphatidylinositide 3-kinases (PI3Ks) play central roles in insulin signal transduction. While the contribution of class Ia PI3K members has been extensively studied, the role of class II members remains poorly understood. The diverse actions of class II PI3K-C2alpha have been attributed to its lipid product PI(3)P. By applying pharmacological inhibitors, transient overexpression and small-interfering RNA-based knockdown of PI3K and PKB/Akt isoforms, together with PI-lipid profiling and live-cell confocal and total internal reflection fluorescence microscopy, we now demonstrate that in response to insulin, PI3K-C2alpha generates PI(3,4)P(2), which allows the selective activation of PKBalpha/Akt1. Knockdown of PI3K-C2alpha expression and subsequent reduction of PKBalpha/Akt1 activity in the pancreatic beta-cell impaired glucose-stimulated insulin release, at least in part, due to reduced glucokinase expression and increased AS160 activity. Hence, our results identify signal transduction via PI3K-C2alpha as a novel pathway whereby insulin activates PKB/Akt and thus discloses PI3K-C2alpha as a potential drugable target in type 2 diabetes. The high degree of codistribution of PI3K-C2alpha and PKBalpha/Akt1 with insulin receptor B type, but not A type, in the same plasma membrane microdomains lends further support to the concept that selectivity in insulin signaling is achieved by the spatial segregation of signaling events.

Several inositol compounds undergo rapid cycles of phosphorylation and dephosphorylation. These cycles are dependent on ATP and energy metabolism. Therefore, interfering with the cellular energy metabolism can change the concentration of rapidly turning over inositols. Many pharmacological inhibitors, apart from their intended action, also affect the energy metabolism of the cells and lower ATP. This can unspecifically influence rapidly turning over inositol phosphates. Thus, the ATP concentration should be checked when reduced inositol phosphates are observed after application of pharmacological inhibitors. A luminescence-based assay for the measurement of ATP and ADP is described. ATP is measured luminometrically using firefly luciferase. Detection of ADP is performed in a two-step enzymatic procedure: (1) The sample ATP is degraded to AMP and (2) ADP is phosphorylated to ATP, which can then be measured luminometrically. This method gives a better signal-to-noise ratio than other methods that do not degrade the sample ATP, but convert ADP directly to ATP and then measure the sum of ATP plus ADP.

Inositides have an important impact on diverse areas of cellular regulation. However, since this area has grown exponentially from the mid 1980s onwards, many workers find themselves relatively new to the field. In this chapter, we establish a broad foundation for the rest of the book by covering some important principles of inositide methodologies. The focus is especially directed to those methods or aspects of methodology not covered in detail in other chapters. This includes the often neglected influence of the inositide precursor, inositol, and important background information relating to the labelling and extraction of inositides from cells and tissues. This introductory section also gives a "birds eye" view of important methods and protocols found within this volume and hopefully acts as a touchstone to assess which of the methodologies described within this book is most appropriate for your particular study(ies) of inositides.

High performance liquid chromatography (HPLC) is an essential analytical tool in the study of the large number of inositol phosphate isomers. This chapter focuses on the separation of inositol polyphosphates from [(3)H]myo-inositol labeled tissues and cells. We review the different HPLC columns that have been used to separate inositol phosphates and their advantages and disadvantages. We describe important elements of sample preparation for effective separations and give examples of how changing factors, such as pH, can considerably improve the resolving ability of the HPLC chromatogram.

By altering the cellular microenvironment and culture media composition, embryonic stem cells (ESCs) can be induced to differentiate in vitro into somatic cell types from the three primitive germ layers. ESC differentiation is regulated by an intricate series of signaling events that result in their transcriptional reprogramming, asymmetric cell division, and differentiation. Using various pharmacological agents and/or genetic manipulations, one can drive and enrich ESC differentiation to specific cell lineages. Identifying the transcriptional fingerprint during ESC differentiation could yield novel targets for genetic or pharmacological regulation to increase the yield of desirable cell types. We discuss here how to culture undifferentiated mouse ESCs (E14 line from 129/Ola) and generate embryoid bodies (EBs). We also discuss in detail how to prepare Affymetrix samples, how to hybridize and scan arrays, and how to quality control data and generate signal values and permutation based P-values.

The stereochemistry of the inositol backbone provides a platform on which to generate a vast array of distinct molecular motifs that are used to convey information both in signal transduction and many other critical areas of cell biology. Diphosphoinositol phosphates, or inositol pyrophosphates, are the most recently characterized members of the inositide family. They represent a new frontier with both novel targets within the cell and novel modes of action. This includes the proposed pyrophosphorylation of a unique subset of proteins. We review recent insights into the structures of these molecules and the properties of the enzymes which regulate their concentration. These enzymes also act independently of their catalytic activity via protein–protein interactions. This unique combination of enzymes and products has an important role in diverse cellular processes including vesicle trafficking, endo- and exocytosis, apoptosis, telomere length regulation, chromatin hyperrecombination, the response to osmotic stress, and elements of nucleolar function.