XPR1 mediates the pancreatic B-cell phosphate flush

Glucose-stimulated insulin secretion is the hallmark of the pancreatic β-cell, a critical player in the regulation of blood glucose concentration. In 1974, the remarkable observation was made that an efflux of intracellular inorganic phosphate (P ) accompanied the events of stimulated insulin secretion. The mechanism behind this "phosphate flush," its association with insulin secretion, and its regulation have since then remained a mystery. We recapitulated the phosphate flush in the MIN6m9 β-cell line and pseudoislets. We demonstrated that knockdown of XPR1, a phosphate transporter present in MIN6m9 cells and pancreatic islets, prevented this flush. Concomitantly, XPR1 silencing led to intracellular P accumulation and a potential impact on Ca signaling. XPR1 knockdown slightly blunted first-phase glucose-stimulated insulin secretion in MIN6m9 cells, but had no significant impact on pseudoislet secretion. In keeping with other cell types, basal P efflux was stimulated by inositol pyrophosphates, and basal intracellular P accumulated following knockdown of inositol hexakisphosphate kinases. However, the glucose-driven phosphate flush occurred despite inositol pyrophosphate depletion. Finally, while it is unlikely that XPR1 directly affects exocytosis, it may protect Ca signaling. Thus, we have revealed XPR1 as the missing mediator of the phosphate flush, shedding light on a 45-year-old mystery

Role of inositol pyrophosphates in pancreatic beta cell function

Inositol pyrophosphates are high energy diphosphate containing molecules that are ubiquitous in eukaryotic cells. They have been implicated in diverse cellular processes ranging from DNA repair, telomere length regulation, ribosome synthesis, cell cycle regulation and apoptosis to osmoregulation, phosphate homeostasis, insulin sensitivity, vesicle trafficking, cytoskeletal rearrangement and exocytosis. The inositol pyrophosphate diphosphoinositol pentakisphosphate (IP7) is present in high levels in pancreatic β-cells. These cells secrete insulin to regulate blood glucose homeostasis. Previous work has shown that IP7 is important for maintaining the immediate exocytotic capacity of β-cells and thus the potential to secrete insulin. However, the physiological regulation and role of IP7 in these cells, especially in response to glucose, remained unexplored. The aims of this Ph.D. work were to investigate the dependence of IP7 on cellular bioenergetic status, the consequent action of IP7 in glucose-induced insulin secretion and IP7’s broader role in cellular regulation. We initially discovered the dependence of IP7 on the cellular ATP/ADP levels in insulin secreting HIT-T15 cells. Off-target reduction in ATP/ADP, upon use of a selection of signal transduction inhibitors, decreased IP7 levels. The compounds tested included inhibitors of phosphatidylinositol 3-kinase, PI3K, (wortmannin, LY294002), phosphatidylinositol 4-kinase, PI4K, (Phenylarsine Oxide, PAO), phospholipase C, PLC, (U73122) and the insulin receptor (HNMPA). We demonstrated for the first time a direct positive correlation between intracellular changes in endogenous ATP/ADP and IP7, pinpointing the regulation of IP7 by the cellular bioenergetic status. This is in agreement with the enzymatic properties of the inositol hexakisphosphate kinases (IP6Ks) that synthesize IP7. Their high Km for ATP makes IP6Ks sensitive to ATP changes. We have also revealed that some inhibitors (PAO, U73122 and LY294002) directly inhibit IP6Ks. We then investigated how physiological changes in ATP/ADP regulate IP7 production in β-cells. Glucose stimulation induced a transient increase in IP7 levels in insulin secreting cell lines and primary islets. Other secretagogues known to increase ATP/ADP, e.g. leucine, also increased IP7 levels. Silencing IP6K1, but not IP6K2, decreased glucose-mediated IP7 production and first phase insulin secretion. Therefore, IP6K1 acts as a key metabolic sensor. In diabetic ob/ob mouse islets the deranged ATP/ADP levels were mirrored by perturbed IP7 production and insulin secretion. Altogether these studies show that metabolic changes in the β-cells are reflected in IP7 levels, which consequently affect exocytosis under both physiological and pathophysiological conditions. IP7 inhibition of Akt/PKB had been described in insulin-sensitive tissues, such as liver, muscle and white fat. β-cells are also regulated by insulin. Therefore, we examined the role of IP7 in modulating the activity of Akt/PKB. To our surprise, the increase in IP7 generated by IP6K1 in glucose-stimulated β-cells was associated with higher Akt/PKB phosphorylation on the T308 and S473 sites. This indicates that IP7 activates Akt/PKB. The results cannot be explained by a direct effect of IP7 on Akt/PKB, because of the inhibitory nature of this interaction. Instead, we propose that Akt/PKB is indirectly activated by IP7 through the IP7-induced increase of insulin secretion and the consequent potentiation of the insulin feedback signaling on β-cell insulin receptors. In conclusion, the dependence of IP7 on cellular bioenergetics status suggests that IP6K1, i.e. the kinase that produces IP7 under glucose stimulation, is a new metabolic sensor in β-cells and is a likely hostage to the disrupted metabolism of type-2 diabetes. The work on Akt/PKB also exposes the complexity of inositol pyrophosphate signaling in different biological settings. Collectively our new findings have considerably broadened the understanding of IP7 regulation and function in β-cells and islets under both physiological and diabetic conditions

Inositol pyrophosphates and Akt/PKB: Is the pancreatic beta-cell the exception to the rule?

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 beta-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 beta-cells. Silencing reduced the glucosestimulated increase in Akt/PKB phosphorylation on T308 and 5473. 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 IP A 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 beta-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 beta-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 beta-cell as in other insulin regulated tissues

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity

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. [Display omitted] •In pancreatic β-cells several inhibitors of signal transduction reduce IP7 levels.•There is a positive correlation between IP7 reduction and decrease in ATP/ADP.•Inhibitors deplete IP7 levels indirectly by decreasing ATP/ADP levels.•Some purportedly specific cell-signaling inhibitors directly target IP6K activity.•Caution is required in interpreting data obtained using inhibitors of inositide metabolism

Inositol hexakisphosphate kinase 1 is a metabolic sensor in pancreatic beta-cells

Diphosphoinositol pentakisphosphate (IP7) is critical for the exocytotic capacity of the pancreatic beta-cell, but its regulation by the primary instigator of beta-cell exocytosis, glucose, is unknown. The high K-m 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 beta-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 beta-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.11Ysciescopu

One-step purification of functional human and rat pancreatic alpha cells

Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca2+ concentration ([Ca2+](i)) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 +/- 1.4%, n = 3 for rat; 95.4 +/- 1.7%, n = 4 for human, mean +/- SEM). These cell fractions showed similar to 5-fold and similar to 4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca2+](i) upon stimulation with L-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 mu M), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat alpha cells, as well as established a novel approach to separate human alpha cells using neither antibodies nor dyes possibly interfering with cellular functions