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Aims/hypothesis: The A (minor) allele of CREBRF rs373863828 has been associated with increased BMI and reduced risk of type 2 diabetes in the Samoan populations of Samoa and American Samoa. Our aim was to test rs373863828 for associations with BMI and the odds of type 2 diabetes, gout and chronic kidney disease (CKD) in Māori and Pacific (Polynesian) people living in Aotearoa/New Zealand., Methods: Linear and logistic regression models were used to analyse the association of the A allele of CREBRF rs373863828 with BMI, log-transformed BMI, waist circumference, type 2 diabetes, gout and CKD in 2286 adults. The primary analyses were adjusted for age, sex, the first four genome-wide principal components and (where appropriate) BMI, waist circumference and type 2 diabetes. The primary analysis was conducted in ancestrally defined groups and association effects were combined using meta-analysis., Results: For the A allele of rs373863828, the effect size was 0.038 (95% CI 0.022, 0.055, p = 4.8 × 10 -6 ) for log-transformed BMI, with OR 0.59 (95% CI 0.47, 0.73, p = 1.9 × 10 -6 ) for type 2 diabetes. There was no evidence for an association of genotype with variance in BMI (p = 0.13), and nor was there evidence for associations with serum urate (β = 0.012 mmol/l, p corrected  = 0.10), gout (OR 1.00, p = 0.98) or CKD (OR 0.91, p = 0.59)., Conclusions/interpretation: Our results in New Zealand Polynesian adults replicate, with very similar effect sizes, the association of the A allele of rs373863828 with higher BMI but lower odds of type 2 diabetes among Samoan adults living in Samoa and American Samoa.

Genetic alterations that potentiate PI3K signaling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report PIK3CA mutation/amplification correlates with poor survival of patients with prostate cancer. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate Pik3ca in mouse prostate epithelium. We show Pik3ca H1047R mutation causes p110α-dependent invasive prostate carcinoma in vivo Furthermore, we report that PIK3CA mutation and PTEN loss coexist in patients with prostate cancer and can cooperate in vivo to accelerate disease progression via AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant Pik3ca mutation and Pten loss caused de novo CRPC. Thus, Pik3ca mutation and Pten deletion are not functionally redundant. Our findings indicate that PIK3CA mutation is an attractive prognostic indicator for prostate cancer that may cooperate with PTEN loss to facilitate CRPC in patients. Significance: We show PIK3CA mutation correlates with poor prostate cancer prognosis and causes prostate cancer in mice. Moreover, PIK3CA mutation and PTEN loss coexist in prostate cancer and can cooperate in vivo to accelerate tumorigenesis and facilitate CRPC. Delineating this synergistic relationship may present new therapeutic/prognostic approaches to overcome castration/PI3K-AKT-mTORC1/2 inhibitor resistance. Cancer Discov; 8(6); 764-79. ©2018 AACR. See related commentary by Triscott and Rubin, p. 682 This article is highlighted in the In This Issue feature, p. 663 ., (©2018 American Association for Cancer Research.)

β-catenin is a multifunctional protein that can act in the canonical Wnt/β-catenin pathway to regulate gene expression but can also bind to cadherin proteins in adherens junctions where it plays a key role in regulating cytoskeleton linked with these junctions. Recently, evidence has been presented indicating an essential role for β-catenin in regulating trafficking of insulin vesicles in β-cells and showing that changes in nutrient levels rapidly alter levels of β-catenin in these cells. Given the importance of neuroendocrine hormone secretion in the regulation of whole body glucose homeostasis, the objective of this study was to investigate whether β-catenin signalling is regulated in the hypothalamus during the normal physiological response to food intake. Rats were subjected to a fasting/re-feeding paradigm, and then samples collected at specific timepoints for analysis of β-catenin expression by immunohistochemistry and Western blotting. Changes in gene expression were assessed by RT-qPCR. Using immunohistochemistry, feeding acutely increased detectable cytoplasmic levels of β-catenin ('stabilized β-catenin') in neurons in specific regions of the hypothalamus involved in metabolic regulation, including the arcuate, dorsomedial and paraventricular nuclei of the hypothalamus. Feeding-induced elevations in β-catenin in these nuclei were associated with increased transcription of several genes that are known to be responsive to Wnt/β-catenin signalling. The effect of feeding was mimicked by administration of the GLP-1 agonist exendin-4, and was characterized by cAMP-dependent phosphorylation of β-catenin at serine residues 552 and 675. The data suggest that β-catenin/TCF signalling is involved in metabolic sensing in the hypothalamus. This article is protected by copyright. All rights reserved., (This article is protected by copyright. All rights reserved.)

GLUT4 is unique among specialized glucose transporters in being exclusively expressed in muscle and adipocytes. In the absence of insulin the distribution of GLUT4 is preferentially intracellular and insulin stimulation results in the movement of GLUT4 containing vesicles to the plasma membrane. This process is responsible for the insulin stimulation of glucose uptake in muscle and fat. While signalling pathways triggering the translocation of GLUT4 are well understood, the mechanisms regulating the intracellular retention of GLUT4 are less well understood. Here we report a role for β-catenin in this process. In 3T3-L1 adipocytes in which β-catenin is depleted, the levels of GLUT4 at and near the plasma membrane rise in unstimulated cells while the subsequent increase in GLUT4 at the plasma membrane upon insulin stimulation is reduced. Small molecule approaches to acutely activate or inhibit β-catenin give results that support the results obtained with siRNA and these changes are accompanied by matching changes in glucose transport into these cells. Together these results indicate that β-catenin is a previously unrecognized regulator of the mechanisms that control the insulin sensitive pool of GLUT4 transporters inside these adipocyte cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In healthy individuals, any rise in blood glucose levels is rapidly countered by the release of insulin from the β-cells of the pancreas which in turn promotes the uptake and storage of the glucose in peripheral tissues. The β-cells possess exquisite mechanisms regulating the secretion of insulin to ensure that the correct amount of insulin is released. These mechanisms involve tight control of the movement of insulin containing secretory vesicles within the β-cells, initially preventing most vesicles being able to move to the plasma membrane. Elevated glucose levels trigger an influx of Ca 2+ that allows fusion of the small number of insulin containing vesicles that are pre-docked at the plasma membrane but glucose also stimulates processes that allow other insulin containing vesicles located further in the cell to move to and fuse with the plasma membrane. The mechanisms controlling these processes are complex and not fully understood but it is clear that the interaction of the β-cells with other β-cells in the islets is very important for their ability to develop the appropriate machinery for proper regulation of insulin secretion. Emerging evidence indicates one factor that is key for this is the formation of homotypic cadherin mediated adherens junctions between β-cells. Here, we review the evidence for this and discuss the mechanisms by which these adherens junctions might regulate insulin vesicle trafficking as well as the implications this has for understanding the dysregulation of insulin secretion seen in pathogenic states., (© 2018 The Author(s).)

Replacement of one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 (1) with sulfonamide containing substituents produced a new class of active and potent PI3Kα inhibitors. Solubility issues prevented all but the 6-amino derivative 17 from being evaluated in vivo, but the clear activity of this compound demonstrated that this class of PI3K inhibitor shows great promise., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Multiple therapeutic agents have been developed to target the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is frequently dysregulated in cancer promoting tumor growth and survival. These include pan PI3K inhibitors, which target class Ia PI3K isoforms and have largely shown limited single agent activity with narrow therapeutic windows in clinical trials. Here, we characterize SN32976, a novel pan PI3K inhibitor, for its biochemical potency against PI3K isoforms and mTOR, kinase selectivity, cellular activity, pharmacokinetics, pharmacodynamics and antitumor efficacy relative to five clinically-evaluated pan PI3K inhibitors: buparlisib, dactolisib, pictilisib, omipalisib and ZSTK474. SN32976 potently inhibited PI3K isoforms and mTOR, displaying preferential activity for PI3Kα and sparing of PI3Kδ relative to the other inhibitors, while showing less off-target activity than the clinical inhibitors in a panel of 442 kinases. The major metabolites of SN32976 were also potent PI3K inhibitors with similar selectivity for PI3Kα as the parent compound. SN32976 compared favorably with the clinically-evaluated PI3K inhibitors in cellular assays, inhibiting pAKT expression and cell proliferation at nM concentrations, and in animal models, inducing a greater extent and duration of pAKT inhibition in tumors than pictilisib, dactolisib and omipalisib at similarly tolerated dose levels and inhibiting tumor growth to a greater extent than dactolisib and ZSTK474 and with similar efficacy to pictilisib and omipalisib. These results suggest that SN32976 is a promising clinical candidate for cancer therapy with enhanced kinase selectivity and preferential inhibition of PI3Kα compared to first generation pan PI3K inhibitors, while retaining comparable anticancer activity.

Phosphoinositide 3-kinases (PI3Ks) are major regulators of many cellular functions, and hyperactivation of PI3K cell signalling pathways is a major target for anticancer drug discovery. PI3Kα is the isoform most implicated in cancer, and our aim is to selectively inhibit this isoform, which may be more beneficial than concurrent inhibition of all Class I PI3Ks. We have used structure-guided design to merge high-selectivity and high-affinity characteristics found in existing compounds. Molecular docking, including the prediction of water-mediated interactions, was used to model interactions between the ligands and the PI3Kα affinity pocket. Inhibition was tested using lipid kinase assays, and active compounds were tested for effects on PI3K cell signalling. The first-generation compounds synthesized had IC 50 (half maximal inhibitory concentration) values >4 μM for PI3Kα yet were selective for PI3Kα over the other Class I isoforms (β, δ and γ). The second-generation compounds explored were predicted to better engage the affinity pocket through direct and water-mediated interactions with the enzyme, and the IC 50 values decreased by ∼30-fold. Cell signalling analysis showed that some of the new PI3Kα inhibitors were more active in the H1047R mutant bearing cell lines SK-OV-3 and T47D, compared with the E545K mutant harbouring MCF-7 cell line. In conclusion, we have used a structure-based design approach to combine features from two different compound classes to create new PI3Kα-selective inhibitors. This provides new insights into the contribution of different chemical units and interactions with different parts of the active site to the selectivity and potency of PI3Kα inhibitors., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Type 2 diabetes (T2D) is a global pandemic. Currently, the drugs used to treat T2D improve hyperglycemic symptom of the disease but the underlying mechanism causing the high blood glucose levels have not been fully resolved. Recently published data showed that salt form of niclosamide improved glucose metabolism in high fat fed mice via mitochondrial uncoupling. However, based on our previous work we hypothesised that niclosamide might also improve glucose metabolism via inhibition of the glucagon signalling in liver in vivo. In this study, mice were fed either a chow or high fat diet containing two different formulations of niclosamide (niclosamide ethanolamine salt - NENS or niclosamide - Nic) for 10 weeks. We identified both forms of niclosamide significantly improved whole body glucose metabolism without altering total body weight or body composition, energy expenditure or insulin secretion or sensitivity. Our study provides evidence that inhibition of the glucagon signalling pathway contributes to the beneficial effects of niclosamide (NENS or Nic) on whole body glucose metabolism. In conclusion, our results suggest that the niclosamide could be a useful adjunctive therapeutic strategy to treat T2D, as hepatic glucose output is elevated in people with T2D and current drugs do not redress this adequately.

The processes regulating glucose-stimulated insulin secretion (GSIS) and its modulation by incretins in pancreatic β-cells are only partly understood. Here we investigate the involvement of β-catenin in these processes. Reducing β-catenin levels using siRNA knockdown attenuated GSIS in a range of β-cell models and blocked the ability of GLP-1 agonists and the depolarizing agent KCl to potentiate this. This could be mimicked in both β-cell models and isolated islets by short-term exposure to the β-catenin inhibitory drug pyrvinium. In addition, short-term treatment with a drug that increases β-catenin levels results in an increase in insulin secretion. The timing of these effects suggests that β-catenin is required for the processes regulating trafficking and/or release of pre-existing insulin granules rather than for those regulated by gene expression. This was supported by the finding that the overexpression of the transcriptional co-activator of β-catenin, transcription factor 7-like 2 (TCF7L2), attenuated insulin secretion, consistent with the extra TCF7L2 translocating β-catenin from the plasma membrane pool to the nucleus. We show that β-catenin depletion disrupts the intracellular actin cytoskeleton, and by using total internal reflectance fluorescence (TIRF) microscopy, we found that β-catenin is required for the glucose- and incretin-induced depletion of insulin vesicles from near the plasma membrane. In conclusion, we find that β-catenin levels modulate Ca 2+ -dependent insulin exocytosis under conditions of glucose, GLP-1, or KCl stimulation through a role in modulating insulin secretory vesicle localization and/or fusion via actin remodeling. These findings also provide insights as to how the overexpression of TCF7L2 may attenuate insulin secretion., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Melanoma, the most aggressive skin cancer type, is responsible for 75% of skin cancer related deaths worldwide. Given that New Zealand (NZ) has the world's highest melanoma incidence, we sought to determine the frequency of mutations in NZ melanomas in recurrently mutated genes. NZ melanomas were from localities distributed between North (35°S-42°S) and South Islands (41°S-47°S). A total of 529 melanomas were analyzed for BRAF exon 15 mutations by Sanger sequencing, and also by Sequenom MelaCarta MassARRAY. While, a relatively low incidence of BRAFV600E mutations (23.4%) was observed overall in NZ melanomas, the incidence of NRAS mutations in South Island melanomas was high compared to North Island melanomas (38.3% vs. 21.9%, P=0.0005), and to The Cancer Genome Atlas database (TCGA) (38.3% vs. 22%, P=0.0004). In contrast, the incidence of EPHB6G404S mutations was 0% in South Island melanomas, and was 7.8% in North Island (P=0.0002). Overall, these data suggest that melanomas from geographically different regions in NZ have markedly different mutation frequencies, in particular in the NRAS and EPHB6 genes, when compared to TCGA or other populations. These data have implications for the causation and treatment of malignant melanoma in NZ., Competing Interests: The authors declare no conflicts of interest.

Cancer stem cells (CSC) exhibit therapy resistance and drive self-renewal of the tumour, making cancer stem cells an important target for therapy. The PI3K signalling pathway has been the focus of considerable research effort, including in glioblastoma (GBM), a cancer that is notoriously resistant to conventional therapy. Different isoforms of the catalytic sub-unit have been associated with proliferation, migration and differentiation in stem cells and cancer stem cells. Blocking these processes in CSC would improve patient outcome. We examined the effect of isoform specific PI3K inhibitors in two models of GBM CSC, an established GBM stem cell line 08/04 and a neurosphere formation model. We identified the dominant catalytic PI3K isoform for each model, and inhibition of the dominant isoform blocked AKT phosphorylation, as did pan-PI3K/mTOR inhibition. Analysis of SOX2, OCT4 and MSI1 expression revealed that inhibition of the dominant p110 subunit increased expression of cancer stem cell genes, while pan-PI3K/mTOR inhibition caused a similar, though not identical, increase in cancer stem cell gene expression. This suggested that PI3K inhibition enhanced, rather than blocked, CSC activity. Careful analysis of the response to specific isoform inhibition will be necessary before specific subunit inhibitors can be successfully deployed against GBM CSC.

Recently, it has been found that glucagon is able to activate the β-catenin signalling pathway leading to increased cyclin D1 and c-Myc expression in liver. Therefore the main aim of the present study is to determine whether the effect of glucagon activating β-catenin signalling leading to increased target gene expression is mediated through cAMP activation of PKA (protein kinase A). Primary rat hepatocytes were incubated with insulin, glucagon or adrenaline (epinephrine) and a range of inhibitors of PI3K (phosphoinositide 3-kinase), Wnt, mitochondrial uncoupler (niclosamide) or PKA inhibitors to dissect out the pathway leading to increased Ser(552) phosphorylation on β-catenin following glucagon exposure. In primary rat hepatocytes, we found that short exposure to glucagon or adrenaline caused a rapid increase in Ser(552) phosphorylation on β-catenin that leads to increased cyclin D1 and c-Myc expression. A range of PI3K and Wnt inhibitors were unable to block the effect of glucagon phosphorylating β-catenin. Interestingly, both niclosamide and the PKA inhibitor H89 blocked the glucagon effect on β-catenin signalling, leading to a reduction in target gene expression. Likewise, niclosamide inhibited cAMP levels and the direct addition of db-cAMP (dibutyryl-cAMP sodium salt) also resulted in Ser(552) phosphorylation of β-catenin. We have identified a new pathway via glucagon signalling that leads to increased β-catenin activity that can be reversed with the antihelminthic drug niclosamide, which has recently shown promise as a potential treatment of T2D (Type 2 diabetes). This novel finding could be useful in liver cancer treatment, particularly in the context of T2D with increased β-catenin activity., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

A novel series of TGX-221 analogues was prepared and tested for their potency against the p110α, p110β, and p110δ isoforms of the PI3K enzyme, and in two cellular assays. The biological results were interpreted in terms of a p110β comparative model, in order to account for their selectivity towards this isoform. A CH2NH type linker is proposed to allow binding into the specificity pocket proposed to accommodate the high p110β-selectivity of TGX-221, although there was limited steric tolerance for substituents on the pendant ring with the 2-position most favourable for substitution., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms (p110α, A66; p110β, TGX-221; p110γ, AS-252424; p110δ, idelalisib), or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.

In the last 20 years the prevalence of metabolic disorders, in particular type 2 diabetes (T2D), has more than doubled. Recently, a strong link between T2D and cancer, in particularly liver cancer has been reported. However, the mechanism connecting the development of type 2 diabetes and cancer remains unknown. One of the biggest drivers of liver cancer is alterations in the Wnt/β-catenin pathway. In this study, we aimed to identify the effect of glucagon on β-catenin in the isolated rat liver. We found glucagon, which is substantially raised in patients with T2D, rapidly phosphorylates β-catenin on serine 552 that is associated with increased expression of genes cyclin D1 (CCND1) and c-Myc (MYC), which are known to be involved in liver cancer. This finding may explain the increased risk of liver cancer in people with T2D.

The second generation antipsychotic drug clozapine is a much more effective therapy for schizophrenia than first generation compounds, but the reasons for this are poorly understood. We have previously shown that one distinguishing feature of clozapine is its ability to raise glucagon levels in animal models and thus causes prolonged hyperinsulinemia without inducing hypoglycaemia. Previous studies have provided evidence that defects in Akt/PKB and GSK3 signalling can contribute to development of psychiatric diseases. Clozapine is known to activate Akt/PKB in the brain, and some studies have indicated that this is due to a direct effect of the drug on the neurons. However, we provide strong evidence that elevated insulin levels induced by clozapine are in fact the real cause of the drug's effects on Akt/PKB and GSK3 in the brain. This suggests that the elevated levels of insulin induced by clozapine may contribute to this drug's therapeutic efficacy.

Second generation antipsychotics cause derangements in glucose metabolism that are often interpreted as insulin resistance. In previous studies we have shown that this is not classical insulin resistance but the drugs were actually inducing a hyperglycaemic state associated with elevated hepatic glucose output (HGO) and increased levels of glucagon and insulin. However, it remains unclear whether these effects are directly elicited by drug actions in the liver and pancreas, or whether they are indirectly mediated. Here we investigated if clozapine is capable of inducing insulin resistance in the liver or enhancing insulin and glucagon secretion from the pancreas. It was observed that insulin signalling was elevated in livers from animals treated with clozapine indicating there was no insulin resistance in the early steps of insulin signalling. To explore whether the defects arise at later stages of insulin action we used an isolated perfused liver system. In this model, clozapine had no direct effect on insulin's counter regulatory effect on epinephrine-induced HGO. In isolated mouse islets clozapine significantly increased glucose-stimulated insulin secretion while simultaneously blocking glucose-induced reductions in glucagon secretion. We also show that the non-peptidic glucagon receptor like peptide-1 (GLP-1) receptor agonist Boc5 was able to overcome the inhibitory effects of clozapine on glucose metabolism. Taken together these results suggest that clozapine does not have any direct effect on glucose metabolism in the liver but it simultaneously stimulates insulin and glucagon secretion, a situation that would allow for the concurrent presence of high glucose and high insulin levels in treated animals., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110β expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110β also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.

The PI3K (phosphoinositide 3-kinase) p110α isoform is activated by oncogenic mutations in many cancers. This has stimulated intense interest in identifying inhibitors of the PI3K pathway as well as p110α-selective inhibitors, and understanding the mechanisms underlying activation by the oncogenic mutations. In the present article, we review recent progress in the structure and function of the p110α enzyme and two of its most common oncogenic mutations, the development of isoform-selective inhibitors, and p110α pharmacology.

The WNT pathway has been well characterized in embryogenesis and tumorigenesis. In humans, specific polymorphisms in the T cell-specific transcription factor 7 and the WNT coreceptor, low-density lipoprotein receptor-related protein-6 (LRP-6), both prominent components of this pathway, correlate with a higher incidence of type 2 diabetes, suggesting that the WNT pathway might be involved in the control of adult glucose homeostasis. We previously demonstrated that glycogen-synthase-kinase-3β (GSK-3β), the key enzyme of the WNT pathway, is increased in the hypothalamus during obesity and exacerbates high-fat diet-induced weight gain as well as glucose intolerance. These data suggest that WNT action in the hypothalamus might be required for normal glucose homeostasis. Here we characterized whether WNT signaling in general is altered in the hypothalamus of adult obese mice relative to controls. First we identified expression of multiple components of this pathway in the murine arcuate nucleus by in situ hybridization. In this region mRNA of ligands and target genes of the WNT pathway were down-regulated in obese and glucose-intolerant leptin-deficient mice. Similarly, the number of cells immunoreactive for the phosphorylated (active) form of the WNT-coreceptor LRP-6 was also decreased in leptin-deficient mice. Leptin treatment normalized expression of the WNT-target genes Axin-2 and Cylin-D1 and increased the number of phospho-LRP-6-immunoreactive cells reaching levels of lean controls. Leptin also increased the levels of phosphorylated (inactive) GSK-3β in the arcuate nucleus, and this effect was colocalized to neuropeptide Y neurons, suggesting that inactivation of GSK-3β may contribute to the neuroendocrine control of energy homeostasis. Taken together our findings identify hypothalamic WNT signaling as an important novel pathway that integrates peripheral information of the body's energy status encoded by leptin.

NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.

The class I phosphatidylinositol 3-kinases (PtdIns3Ks) mediate the effects of many hormones and growth factors on a wide range of cellular processes, and activating mutations or gene amplifications of class I PtdIns3K isoforms are known to contribute to oncogenic processes in a range of tumours. Consequently, a number of small-molecule PtdIns3K inhibitors are under development and in clinical trial. The central signalling role of PtdIns3K in many cellular processes suggests there will be on-target side effects associated with the use of these agents. To gain insights into what these might be we investigated the effect of extended daily dosing of eight small-molecule inhibitors of class Ia PtdIns3Ks. Animals were characterized in metabolic cages to analyse food intake, oxygen consumption and movement. Insulin tolerance and body composition were analysed at the end of the experiment, the latter using EchoMRI. Bone volume and strength was assessed by micro-CT and three-point bending, respectively. Surprisingly, after sustained dosing with pan-PtdIns3K inhibitors and selective inhibitors of the p110α isoform there was a resolution of the impairments in insulin tolerance observed in drug-naïve animals treated with the same drugs. However, pan-PtdIns3K inhibitors and selective inhibitors of the p110α have deleterious effects on animal growth, animal behaviour and bone volume and strength. Together, these findings identify a range of on target effects of PtdIns3K inhibitors and suggest use of these drugs in humans may have important adverse effects on metabolism, body composition, behaviour and skeletal health., (© 2013 FEBS.)

PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.

Adiponectin is an adipokine that can suppress the proliferation of various human carcinoma cells. Although its anti-tumor activities have been suggested by many clinical investigations and animal studies, the underlying mechanisms are not fully characterized. In MMTV-polyomavirus middle T antigen (MMTV-PyVT) transgenic mice models, reduced- or complete loss-of-adiponectin expression promotes mammary tumor development. The present study demonstrated that while tumor development in control MMTV-PyVT mice is associated with a progressively decreased circulating cholesterol concentration, adiponectin deficient MMTV-PyVT mice showed significantly elevated total- and low density lipoprotein (LDL)-cholesterol levels. Cholesterol contents in tumors derived from adiponectin deficient mice were dramatically augmented. High fat high cholesterol diet further accelerated the tumor development in adiponectin deficient PyVT mice. The protein levels of LDL receptor (LDLR) were found to be upregulated in adiponectin-deficient tumor cells. In human breast carcinoma cells, treatment with LDL-cholesterol or overexpressing LDLR elevates nuclear beta-catenin activity and facilitates tumor cell proliferation. On the other hand, adiponectin decreased LDLR protein expression in breast cancer cells and inhibited LDL-cholesterol-induced tumor cell proliferation. Both in vivo and in vitro evidence demonstrated a stimulatory effect of adiponectin on autophagy process, which mediated the down-regulation of LDLR. Adiponectin-induced reduction of LDLR was blocked by treatment with a specific inhibitor of autophagy, 3-methyladenine. In conclusion, the study demonstrates that adiponectin elicits tumor suppressive effects by modulating cholesterol homeostasis and LDLR expression in breast cancer cells, which is at least in part attributed to its role in promoting autophagic flux.

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 βc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 βc receptor (βic). We find p110α, p110β and p110γ all phosphorylate βic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards βic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn(2+) dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110β and p110γ all display significant activity in the presence of Mg(2+). Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.

A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Pancreatic β-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the β-catenin signalling pathway plays an important role in regulating β-cell function, but the mechanisms regulating β-catenin signalling in these cells is not well understood. In the present study we show that β-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and β-TC6-F7 β-cell models. We found a glucose-dependent increase in levels of β-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of β-catenin. This was associated with an increase in phosphorylation of β-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on β-catenin signalling. Finally, reducing β-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate β-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in β-cells.

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer., Competing Interests: The authors have declared that no competing interests exist.

Synergistic activation by heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110β from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110β is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110β-dependent functions, we identified the site in p110β that binds to the Gβγ subunit of G proteins. Mutation of this site eliminated Gβγ-dependent activation of PI3Kβ (a dimer of p110β and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide-mediated activation. Disrupting the p110β-Gβγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kβ in fibroblasts and reduced the proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kβ could provide a therapeutic approach for tumors that depend on p110β for growth and metastasis.

GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lep(ob/ob) mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.

Transforming growth factor (TGF) β1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K) is one of the signaling pathways implicated in TGFβ1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFβ1 induced pro-inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFβ1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110β inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL-6, calponin or smooth muscle α-actin (α-SMA) expression. However, TGFβ1 increased calponin and α-SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFβ1 induced IL-6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFβ1 induced production of the contractile proteins calponin and α-SMA and the proinflammatory cytokine IL-6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling., (Copyright © 2011 Wiley Periodicals, Inc.)

The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110β isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser(773), A-66S bound the region 1 non-conserved amino acid p110α Gln(859), and J-32 binding had an indirect interaction with Lys(776) and Ile(771). The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.

We determined myofibrillar and mitochondrial protein fractional synthesis rates (FSR), intramuscular signaling protein phosphorylation, and mRNA expression responses after isolated bouts of resistance exercise (RE), aerobic exercise (AE), or in combination [termed concurrent exercise (CE)] in sedentary middle-aged men. Eight subjects (age = 53.3 ± 1.8 yr; body mass index = 29.4 ± 1.4 kg·m(2)) randomly completed 8 × 8 leg extension repetitions at 70% of one repetition-maximum, 40 min of cycling at 55% peak aerobic power output (AE), or (consecutively) 50% of the RE and AE trials (CE). Biopsies were obtained (during a primed, constant infusion of l-[ring-(13)C(6)]phenylalanine) while fasted, and at 1 and 4 h following postexercise ingestion of 20 g of protein. All trials increased mitochondrial FSR above fasted rates (RE = 1.3-fold; AE = 1.5; CE = 1.4; P < 0.05), although only CE (2.2) and RE (1.8) increased myofibrillar FSR (P < 0.05). At 1 h postexercise, phosphorylation of Akt on Ser(473) (CE = 7.7; RE = 4.6) and Thr(308) (CE = 4.4; RE = 2.9), and PRAS40 on Thr(246) (CE = 3.8; AE = 2.5) increased (P < 0.05), with CE greater than AE for Akt Ser(473)-Thr(308) and greater than RE for PRAS40 (P < 0.05). Despite increased phosphorylation of Akt-PRAS40, phosphorylation of mammalian target of rapamycin (Ser(2448)) remained unchanged (P > 0.05), while rpS6 (Ser(235/236)) increased only in RE (10.4) (P < 0.05). CE and AE both resulted in increased peroxisome proliferator receptor-γ coactivator 1-α (PGC1α) expression at 1 h (CE = 2.9; AE = 2.8; P < 0.05) and 4 h (CE = 2.6; AE = 2.4) and PGC1β expression at 4 h (CE = 2.1; AE = 2.6; P < 0.05). These data suggest that CE-induced acute stimulation of myofibrillar and mitochondrial FSR, protein signaling, and mRNA expression are equivalent to either isolate mode (RE or AE). These results occurred without an interference effect on muscle protein subfractional synthesis rates, protein signaling, or mRNA expression.

Although it has been known for some time that PTEN-null tumors require expression of the p110β isoform of phosphoinositide 3-kinase for growth, the corollary demonstration that small-molecule inhibitors of p110β are effective drugs for such tumors has not been shown. This has now been rectified by the demonstration that the TGX221 analogue KIN-193 is effective in mouse xenografts of HCC70 and PC3 human tumor cell lines., (© 2012 AACR.)

The flavone acetic acid derivative DMXAA [5,6-dimethylXAA (xanthenone-4-acetic acid), Vadimezan, ASA404] is a drug that displayed vascular-disrupting activity and induced haemorrhagic necrosis and tumour regression in pre-clinical animal models. Both immune-mediated and non-immune-mediated effects contributed to the tumour regression. The vascular disruption was less in human tumours, with immune-mediated effects being less prominent, but nonetheless DMXAA showed promising effects in Phase II clinical trials in non-small-cell lung cancer. However, these effects were not replicated in Phase III clinical trials. It has been difficult to understand the differences between the pre-clinical findings and the later clinical trials as the molecular targets for the agent have never been clearly established. To investigate the mechanism of action, we sought to determine whether DMXAA might target protein kinases. We found that, at concentrations achieved in blood during clinical trials, DMXAA has inhibitory effects against several kinases, with most potent effects being on members of the VEGFR (vascular endothelial growth factor receptor) tyrosine kinase family. Some analogues of DMXAA were even more effective inhibitors of these kinases, in particular 2-MeXAA (2-methylXAA) and 6-MeXAA (6-methylXAA). The inhibitory effects were greatest against VEGFR2 and, consistent with this, we found that DMXAA, 2-MeXAA and 6-MeXAA were able to block angiogenesis in zebrafish embryos and also inhibit VEGFR2 signalling in HUVECs (human umbilical vein endothelial cells). Taken together, these results indicate that at least part of the effects of DMXAA are due to it acting as a multi-kinase inhibitor and that the anti-VEGFR activity in particular may contribute to the non-immune-mediated effects of DMXAA on the vasculature.

Background: The phosphatidylinositol-3-kinase (PI3K-PKB), mitogen activated protein kinase (MEK-ERK) and the mammalian target of rapamycin (mTOR- p70S6K), are thought to regulate many aspects of tumour cell proliferation and survival. We have examined the utilisation of these three signalling pathways in a number of cell lines derived from patients with metastatic malignant melanoma of known PIK3CA, PTEN, NRAS and BRAF mutational status., Methods: Western blotting was used to compare the phosphorylation status of components of the PI3K-PKB, MEK-ERK and mTOR-p70S6K signalling pathways, as indices of pathway utilisation., Results: Normal melanocytes could not be distinguished from melanoma cells on the basis of pathway utilisation when grown in the presence of serum, but could be distinguished upon serum starvation, where signalling protein phosphorylation was generally abrogated. Surprisingly, the differential utilisation of individual pathways was not consistently associated with the presence of an oncogenic or tumour suppressor mutation of genes in these pathways., Conclusion: Utilisation of the PI3K-PKB, MEK-ERK and mTOR-p70S6K signalling pathways in melanoma, as determined by phosphorylation of signalling components, varies widely across a series of cell lines, and does not directly reflect mutation of genes coding these components. The main difference between cultured normal melanocytes and melanoma cells is not the pathway utilisation itself, but rather in the serum dependence of pathway utilisation.

In the present study, we have examined whether IKKβ [IκB (inhibitor of nuclear factor κB) kinase β] plays a role in feedback inhibition of the insulin signalling cascade. Insulin induces the phosphorylation of IKKβ, in vitro and in vivo, and this effect is dependent on intact signalling via PI3K (phosphoinositide 3-kinase), but not PKB (protein kinase B). To test the hypothesis that insulin activates IKKβ as a means of negative feedback, we employed a variety of experimental approaches. First, pharmacological inhibition of IKKβ via BMS-345541 did not potentiate insulin-induced IRS1 (insulin receptor substrate 1) tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKβ target sites. Secondly, adenovirus-mediated overexpression of wild-type IKKβ in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, IRS1 tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signalling was not potentiated in mouse embryonic fibroblasts lacking IKKβ. Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKβ to IRS1, supporting our findings that IKKβ, although activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin signalling cascade.

In in vitro studies class-I PI3Ks (phosphoinositide 3-kinases), class-II PI3Ks and mTOR (mammalian target of rapamycin) have all been described as having roles in the regulation of glucose metabolism. The relative role each plays in the normal signalling processes regulating glucose metabolism in vivo is less clear. Knockout and knockin mouse models have provided some evidence that the class-I PI3K isoforms p110α, p110β, and to a lesser extent p110γ, are necessary for processes regulating glucose metabolism and appetite. However, in these models the PI3K activity is chronically reduced. Therefore we analysed the effects of acutely inhibiting PI3K isoforms alone, or PI3K and mTOR, on glucose metabolism and food intake. In the present study impairments in glucose tolerance, insulin tolerance and increased hepatic glucose output were observed in mice treated with the pan-PI3K/mTOR inhibitors PI-103 and NVP-BEZ235. The finding that ZSTK474 has similar effects indicates that these effects are due to inhibition of PI3K rather than mTOR. The p110α-selective inhibitors PIK75 and A66 also induced these phenotypes, but inhibitors of p110β, p110δ or p110γ induced only minor effects. These drugs caused no significant effects on BMR (basal metabolic rate), O2 consumption or water intake, but BEZ235, PI-103 and PIK75 did cause a small reduction in food consumption. Surprisingly, pan-PI3K inhibitors or p110α inhibitors caused reductions in animal movement, although the cause of this is not clear. Taken together these studies provide pharmacological evidence to support a pre-eminent role for the p110α isoform of PI3K in pathways acutely regulating glucose metabolism.

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A structure-activity relationship (SAR) study of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110β, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1(-/-) mice, and at a dose of 50 mg/kg given by ip injection at a qd × 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.

The atypical antipsychotic drug olanzapine induces weight gain and defects in glucose metabolism in patients. Using a rat model we investigated the effects of acute and long term olanzapine treatment on weight gain, food preference and glucose metabolism. Olanzapine treated rats fed a chow diet grew more slowly than vehicle controls but olanzapine treated animals fed a high fat/sugar diet grew faster than control animals on the same diet. These changes in weight were paralleled by changes in fat mass. Olanzapine also induced a strong preference for a high fat/high sugar diet. Acute exposure to olanzapine rapidly induced severe impairments of glucose tolerance and increased insulin secretion but did not impair insulin tolerance. These results indicate the defect in glucose metabolism induced by acute olanzapine treatment was most likely due to increased hepatic glucose output associated with a reduction in active GLP-1 levels and correspondingly high glucagon levels.

Maintenance of cardiac structure and Z-disc signaling are key factors responsible for protecting the heart in a setting of stress, but how these processes are regulated is not well defined. We recently demonstrated that PI3K(p110α) protects the heart against myocardial infarction. The aim of this study was to determine whether PI3K(p110α) directly regulates components of the Z-disc and cardiac structure. To address this question, a unique three-dimensional virtual muscle model was applied to gene expression data from transgenic mice with increased or decreased PI3K(p110α) activity under basal conditions (sham) and in a setting of myocardial infarction to display the location of structural proteins. Key findings from this analysis were then validated experimentally. The three-dimensional virtual muscle model visually highlighted reciprocally regulated transcripts associated with PI3K activation that encoded key components of the Z-disc and costamere, including melusin. Studies were performed to assess whether PI3K and melusin interact in the heart. Here, we identify a novel melusin-PI3K interaction that generates lipid kinase activity. The direct impact of PI3K(p110α) on myocyte structure was assessed by treating neonatal rat ventricular myocytes with PI3K(p110α) inhibitors and examining the myofiber morphology of hearts from PI3K transgenic mice. Results demonstrate that PI3K is critical for myofiber maturation and Z-disc alignment. In summary, PI3K regulates the expression of genes essential for cardiac structure and Z-disc signaling, interacts with melusin, and is critical for Z-disc alignment.

Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110α in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110α have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110α/p110β/p110δ in all cell lines tested. However, inhibition of p110α alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110α and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110α inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110α alone has the potential to block growth factor signalling and reduce growth in a subset of tumours., (© 2011 The Author(s))

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in the airway remodeling associated with asthma. The class IA PI3K isoforms are known to be activated by growth factors and cytokines. Because this pathway is a possible site of pharmacological intervention for treating the disease, it is important to know which isoforms contribute to this process. Therefore, we used a pharmacological approach to investigate the roles of the three class IA PI3K isoforms (p110α, p110β, and p110δ) in airway remodeling using airway smooth muscle (ASM) cells derived from asthmatic subjects and ASM cells and lung fibroblasts from nonasthmatic subjects. These studies used the inhibitors N'-[(E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylidene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide (PIK75) (which selectively inhibits p110α), 7-methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one (TGX221) (which selectively inhibits p110β), and 2-[(6-amino-9H-purin-9-yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (IC87114) (which selectively inhibits p110δ). Cells were stimulated with transforming growth factor-β (TGFβ) and/or 10% fetal bovine serum in the presence or absence of inhibitor or vehicle control (dimethyl sulfoxide). PIK75, but not TGX221 or IC87114, attenuated TGFβ-induced fibronectin deposition in all cell types tested. PIK75 and TGX221 each decreased secretion of vascular endothelial growth factor and interleukin-6 in nonasthmatic ASM cells and lung fibroblasts, whereas TGX221 was not as effective in asthmatic ASM cells. In addition, PIK75 decreased cell survival in TGFβ-stimulated asthmatic, but not nonasthmatic, ASM cells. In conclusion, specific PI3K isoforms may play a role in pathophysiological events relevant to airway wall remodeling.

Background: Fracture risk is increased in patients with schizophrenia, who often receive long-term therapy with anti-psychotic drugs. The mechanisms by which skeletal fragility is increased in patients with psychosis include increased risk of falling, but direct skeletal toxicity of anti-psychotic drugs is a possibility that has not been investigated., Methods: We examined the skeletal effects, in vivo and in vitro, of a typical anti-psychotic drug, haloperidol, which primarily inhibits dopaminergic signaling, and an atypical anti-psychotic drug, clozapine, which predominantly inhibits serotonergic signaling., Results: In growing rats, 42 days of clozapine treatment reduced whole body bone mineral density by 15% (P<0.01 vs vehicle), and trabecular and cortical bone volume, as assessed by microcomputed tomography, by 29% and 15%, respectively (P<0.05 vs vehicle for each). Treatment with haloperidol did not affect bone density. Clozapine, but not haloperidol, transiently increased levels of serum corticosterone, and decreased levels of serum testosterone. In vitro, clozapine dose-dependently decreased osteoblast mitogenesis, osteoblast differentiation and osteoclastogenesis, while haloperidol did not affect any of these parameters., Conclusions: These data demonstrate that clozapine, but not haloperidol, exerts adverse skeletal effects in rodents, and that this effect may be attributable to direct actions to reduce osteoblast growth and function. Long-term administration of clozapine may therefore negatively affect bone health, and clinical studies to investigate this possibility are warranted., (Copyright © 2010 Elsevier B.V. All rights reserved.)