Dominant Role of PI3K p110α over p110ß in Insulin and ß-Adrenergic Receptor Signalling.

Attribution of specific roles to the two ubiquitously expressed PI 3-kinase (PI3K) isoforms p110α and p110ß in biological functions they have been implicated, such as in insulin signalling, has been challenging. While p110α has been demonstrated to be the principal isoform activated downstream of the insulin receptor, several studies have provided evidence for a role of p110ß. Here we have used isoform-selective inhibitors to estimate the relative contribution of each of these isoforms in insulin signalling in adipocytes, which are a cell type with essential roles in regulation of metabolism at the systemic level. Consistent with previous genetic and pharmacological studies, we found that p110α is the principal isoform activated downstream of the insulin receptor under physiological conditions. p110α interaction with Ras enhanced the strength of p110α activation by insulin. However, this interaction did not account for the selectivity for p110α over p110ß in insulin signalling. We also demonstrate that p110α is the principal isoform activated downstream of the ß-adrenergic receptor (ß-AR), another important signalling pathway in metabolic regulation, through a mechanism involving activation of the cAMP effector molecule EPAC1. This study offers further insights in the role of PI3K isoforms in the regulation of energy metabolism with implications for the therapeutic application of selective inhibitors of these isoforms.

C. elegans feed yolk to their young in a form of primitive lactation.

The nematode Caenorhabditis elegans exhibits rapid senescence that is promoted by the insulin/IGF-1 signalling (IIS) pathway via regulated processes that are poorly understood. IIS also promotes production of yolk for egg provisioning, which in post-reproductive animals continues in an apparently futile fashion, supported by destructive repurposing of intestinal biomass that contributes to senescence. Here we show that post-reproductive mothers vent yolk which can be consumed by larvae and promotes their growth. This implies that later yolk production is not futile; instead vented yolk functions similarly to milk. Moreover, yolk venting is promoted by IIS. These findings suggest that a self-destructive, lactation-like process effects resource transfer from postreproductive C. elegans mothers to offspring, in a fashion reminiscent of semelparous organisms that reproduce in a single, suicidal burst. That this process is promoted by IIS provides insights into how and why IIS shortens lifespan in C. elegans.

Publisher Correction: Enhanced ß-adrenergic signalling underlies an age-dependent beneficial metabolic effect of PI3K p110α inactivation in adipose tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increased mitochondrial and lipid metabolism is a conserved effect of Insulin/PI3K pathway downregulation in adipose tissue.

The Insulin/IGF-1 signalling (IIS) pathway plays an essential role in the regulation of glucose and lipid homeostasis. At the same time, a reduction in the IIS pathway activity can extend lifespan and healthspan in various model organisms. Amongst a number of body organs that sense and respond to insulin/IGF-1, the adipose tissue has a central role in both the metabolic and lifespan effects of IIS at the organismal level. Genetic inactivation of IIS components specifically in the adipose tissue has been shown before to improve metabolic profile and extend lifespan in various model organisms. We sought to identify conserved molecular mechanisms that may underlie the beneficial effects of IIS inhibition in the adipose tissue, specifically at the level of phosphoinositide 3-kinase (PI3K), a key IIS effector molecule. To this end, we inactivated PI3K by genetic means in the fly fat body and by pharmacological inhibition in mammalian adipocytes. Gene expression studies revealed changes to metabolism and upregulation of mitochondrial activity in mouse adipocytes and fly fat bodies with downregulated PI3K, which were confirmed by biochemical assays in mammalian adipocytes. These data suggest that PI3K inactivation has a conserved effect of upregulating mitochondrial metabolism in both fly and mammalian adipose tissue, which likely contributes to the health- and life-span extending effect of IIS pathway downregulation.

Enhanced ß-adrenergic signalling underlies an age-dependent beneficial metabolic effect of PI3K p110α inactivation in adipose tissue.

The insulin/IGF-1 signalling pathway is a key regulator of metabolism and the rate of ageing. We previously documented that systemic inactivation of phosphoinositide 3-kinase (PI3K) p110α, the principal PI3K isoform that positively regulates insulin signalling, results in a beneficial metabolic effect in aged mice. Here we demonstrate that deletion of p110α specifically in the adipose tissue leads to less fat accumulation over a significant part of adult life and allows the maintenance of normal glucose tolerance despite insulin resistance. This effect of p110α inactivation is due to a potentiating effect on ß-adrenergic signalling, which leads to increased catecholamine-induced energy expenditure in the adipose tissue. Our findings provide a paradigm of how partial inactivation of an essential component of the insulin signalling pathway can have an overall beneficial metabolic effect and suggest that PI3K inhibition could potentiate the effect of ß-adrenergic agonists in the treatment of obesity and its associated comorbidities.

Growth factor, energy and nutrient sensing signalling pathways in metabolic ageing.

The field of the biology of ageing has received increasing attention from a biomedical point of view over the past decades. The main reason has been the realisation that increases in human population life expectancy are accompanied by late onset diseases. Indeed, ageing is the most important risk factor for a number of neoplastic, neurodegenerative and metabolic pathologies. Advances in the knowledge of the genetics of ageing, mainly through research in model organisms, have implicated various cellular processes and the respective signalling pathways that regulate them in cellular and organismal ageing. Associated with ageing is a dysregulation of metabolic homeostasis usually manifested as age-related obesity, diminished insulin sensitivity and impaired glucose and lipid homeostasis. Metabolic deterioration contributes to the ageing phenotype and metabolic pathologies are thought to be one of the main factors limiting the potential for lifespan extension. Great efforts have been directed towards identifying pharmacological interventions with the potential to improve healthspan and a number of natural and synthetic compounds have shown promise in achieving beneficial metabolic effects.

Insulin resistance uncoupled from dyslipidemia due to C-terminal PIK3R1 mutations.

Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85α/p55α/p50α subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85α with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85α overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome.

Inactivation of class II PI3K-C2α induces leptin resistance, age-dependent insulin resistance and obesity in male mice.

AIMS/HYPOTHESIS: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2ß and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2ß increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice. METHODS: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a). RESULTS: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age. CONCLUSIONS/INTERPRETATION: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis. ACCESS TO RESEARCH MATERIALS: All reagents are available upon request.

Long-term p110α PI3K inactivation exerts a beneficial effect on metabolism.

The insulin/insulin-like growth factor-1 signalling (IIS) pathway regulates cellular and organismal metabolism and controls the rate of aging. Gain-of-function mutations in p110α, the principal mammalian IIS-responsive isoform of PI 3-kinase (PI3K), promote cancer. In contrast, loss-of-function mutations in p110α impair insulin signalling and cause insulin resistance, inducing a pre-diabetic state. It remains unknown if long-term p110α inactivation induces further metabolic deterioration over time, leading to overt unsustainable pathology. Surprisingly, we find that chronic p110α partial inactivation in mice protects from age-related reduction in insulin sensitivity, glucose tolerance and fat accumulation, and extends the lifespan of male mice. This beneficial effect of p110α inactivation derives in part from a suppressed down-regulation of insulin receptor substrate (IRS) protein levels induced by age-related hyperinsulinemia, and correlates with enhanced insulin-induced Akt signalling in aged p110α-deficient mice. This temporal metabolic plasticity upon p110α inactivation indicates that prolonged PI3K inhibition, as intended in human cancer treatment, might not negatively impact on organismal metabolism.

Developmental defects and rescue from glucose intolerance of a catalytically-inactive novel Ship2 mutant mouse.

The function of the phosphoinositide 5-phosphatase Ship2 was investigated in a new mouse model expressing a germline catalytically-inactive Ship2(∆/∆) mutant protein. Ship2(∆/∆) mice were viable with defects in somatic growth and in development of muscle, adipose tissue and female genital tract. Lipid metabolism and insulin secretion were also affected in these mice, but glucose tolerance, insulin sensitivity and insulin-induced PKB phosphorylation were not. We expected that the expression of the catalytically inactive Ship2 protein in PI 3'-kinase-defective p110α(D933A/+) mice would counterbalance the phenotypes of parental mice by restoring normal PKB signaling but, for most of the parameters tested, this was not the case. Indeed, often, the Ship2(∆/∆) phenotype had a dominant effect over the p110α(D933A/+) phenotype and, sometimes, there was a surprising additive effect of both mutations. p110α(D933A/+)Ship2(∆/∆) mice still displayed a reduced PKB phosphorylation in response to insulin, compared to wild type mice yet had a normal glucose tolerance and insulin sensitivity, like the Ship2(∆/∆) mice. Together, our results suggest that the Ship2(∆/∆) phenotype is not dependent on an overstimulated class I PI 3-kinase-PKB signaling pathway and thus, indirectly, that it may be more dependent on the lack of Ship2-produced phosphatidylinositol 3,4-bisphosphate and derived phosphoinositides.

Activity of any class IA PI3K isoform can sustain cell proliferation and survival.

Small molecule inhibitors of PI3K for oncology mainly target the class I PI3Ks, comprising the p110alpha, beta, gamma, and delta isoforms, of which only p110alpha is mutated in cancer. To assess the roles of class I PI3K isoforms in cell proliferation and survival, we generated immortalized mouse leukocyte and fibroblast models in which class I PI3Ks were inactivated by genetic and pharmacological approaches. In IL3-dependent hemopoietic progenitor cells (which express all four class I PI3K isoforms), genetic inactivation of either p110alpha or p110delta did not affect cell proliferation or survival or sensitize to p110beta or p110gamma inactivation. Upon compound inactivation of p110alpha and p110delta, which removed >90% of p85-associated PI3K activity, remarkably, cells continued to proliferate effectively, with p110beta assuming an essential role in signaling and cell survival. Furthermore, under these conditions of diminished class I PI3K activity, input from the ERK pathway became important for cell survival. Similar observations were made in mouse embryonic fibroblasts (which mainly express p110alpha and p110beta) in which p110alpha or p110beta could sustain cell proliferation as a single isoform. Taken together, these data demonstrate that a small fraction of total class I PI3K activity is sufficient to sustain cell survival and proliferation. Persistent inhibition of selected PI3K isoforms can allow the remaining isoform(s) to couple to upstream signaling pathways in which they are not normally engaged. Such functional redundancy of class IA PI3K isoforms upon sustained PI3K inhibition has implications for the development and use of PI3K inhibitors in cancer.

Phosphoinositide signalling pathways in metabolic regulation.

The Insulin Receptor/PI 3-kinase (INSR/PI3K) signalling pathway is a key regulator of cell and organismal metabolism. Phosphoinositides generated by PI 3-kinases following insulin and other metabolic hormone receptor activation give rise to signalling cascades involving a multitude of effector molecules. The physiological roles of these molecules have been dissected with the use of both pharmacological and genetic tools. Furthermore, tissue-specific mutagenesis has revealed the extent to which individual insulin-target organs and signalling molecules contribute to whole-body carbohydrate and lipid homeostasis. These studies have generated important information with respect to the function of these molecules in normal physiology and their implication in the development of metabolic diseases such as type-2 diabetes and obesity.

Angiogenesis selectively requires the p110alpha isoform of PI3K to control endothelial cell migration.

Phosphoinositide 3-kinases (PI3Ks) signal downstream of multiple cell-surface receptor types. Class IA PI3K isoforms couple to tyrosine kinases and consist of a p110 catalytic subunit (p110alpha, p110beta or p110delta), constitutively bound to one of five distinct p85 regulatory subunits. PI3Ks have been implicated in angiogenesis, but little is known about potential selectivity among the PI3K isoforms and their mechanism of action in endothelial cells during angiogenesis in vivo. Here we show that only p110alpha activity is essential for vascular development. Ubiquitous or endothelial cell-specific inactivation of p110alpha led to embryonic lethality at mid-gestation because of severe defects in angiogenic sprouting and vascular remodelling. p110alpha exerts this critical endothelial cell-autonomous function by regulating endothelial cell migration through the small GTPase RhoA. p110alpha activity is particularly high in endothelial cells and preferentially induced by tyrosine kinase ligands (such as vascular endothelial growth factor (VEGF)-A). In contrast, p110beta in endothelial cells signals downstream of G-protein-coupled receptor (GPCR) ligands such as SDF-1alpha, whereas p110delta is expressed at low level and contributes only minimally to PI3K activity in endothelial cells. These results provide the first in vivo evidence for p110-isoform selectivity in endothelial PI3K signalling during angiogenesis.

Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation.

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.

Signalling by PI3K isoforms: insights from gene-targeted mice.

Phosphoinositide 3-kinases (PI3Ks) generate lipids that control a wide variety of intracellular signalling pathways. Part of this diversity in PI3K actions stems from the broad range of protein effectors of the PI3K lipids. A further layer of complexity is added by the existence of multiple isoforms of PI3K. Gene-targeting studies in the mouse have recently uncovered key roles for specific PI3K isoforms in immunity, metabolism and cardiac function. Remarkably, some of these actions do not require PI3K catalytic activity. In addition, loss-of-expression of certain PI3K genes leads to increased PI3K signalling following insulin stimulation. PI3K gene targeting has, in many cases, led to altered expression of the non-targeted PI3K subunits, making it difficult to exclude that some of the reported phenotypes result from 'knock-on' effects of PI3K gene deletion. Targeting strategies that take into account the complex interplay between members of the PI3K family will be crucial to gain a full understanding of the physiological roles of the isoforms of PI3K.

eIF4E binding protein 1 and H-Ras are novel substrates for the protein kinase activity of class-I phosphoinositide 3-kinase.

Class-I phosphoinositide 3-kinases (PI 3-kinases) are dual specificity enzymes that possess both lipid and protein kinase activity. While the best characterized property of this protein kinase is as an autokinase activity, there have also been reports it can phosphorylate exogenous substrates including peptides, IRS-1 and PDE-3B. The identification of two novel potential protein substrates of PI 3-kinase is described here. By employing in vitro kinase assays using recombinant proteins as the substrates, it is shown that the translational regulator 4EBP1 becomes phosphorylated by the p110alpha and p110gamma isoforms of class-I PI 3-kinases. The lipid kinase activity of both these isoforms is increased by allosteric binding of H-Ras or betagamma subunits of heterotrimeric G proteins, but we find this is not the case for the protein kinase activity. Surprisingly though, a site on H-Ras is phosphorylated by p110alpha and p110gamma. This raises the possibility that these proteins could serve as physiological substrates for the protein kinase activity of PI 3-kinase and suggests this activity operates in a physiological context by phosphorylating substrates other than the PI 3-kinase itself. This may be particularly important in regulating the interaction of Ras with PI 3-kinase.

Regulation of phosphoinositide 3-kinase by its intrinsic serine kinase activity in vivo.

One potentially important mechanism for regulating class Ia phosphoinositide 3-kinase (PI 3-kinase) activity is autophosphorylation of the p85 alpha adapter subunit on Ser608 by the intrinsic protein kinase activity of the p110 catalytic subunit, as this downregulates the lipid kinase activity in vitro. Here we investigate whether this phosphorylation can occur in vivo. We find that p110 alpha phosphorylates p85 alpha Ser608 in vivo with significant stoichiometry. However, p110 beta is far less efficient at phosphorylating p85 alpha Ser608, identifying a potential difference in the mechanisms by which these two isoforms are regulated. The p85 alpha Ser608 phosphorylation was increased by treatment with insulin, platelet-derived growth factor, and the phosphatase inhibitor okadaic acid. The functional effects of this phosphorylation are highlighted by mutation of Ser608, which results in reduced lipid kinase activity and reduced association of the p110 alpha catalytic subunit with p85 alpha. The importance of this phosphorylation was further highlighted by the finding that autophosphorylation on Ser608 was impaired, while lipid kinase activity was increased, in a p85 alpha mutant recently discovered in human tumors. These results provide the first evidence that phosphorylation of Ser608 plays a role as a shutoff switch in growth factor signaling and contributes to the differences in functional properties of different PI 3-kinase isoforms in vivo.

Direct interaction of major histocompatibility complex class II-derived peptides with class Ia phosphoinositide 3-kinase results in dose-dependent stimulatory effects.

Peptides corresponding to residues 65-79 of human lymphocyte antigen class II sequence (DQA*03011) are cell-permeable and at high concentrations block activation of protein kinase B/Akt and p70-S6 kinase in T-cells, effects attributed to inhibition of phosphoinositide (PI) 3-kinase activity. To understand the molecular basis of this, we analyzed the effect this peptide had on activity of class I PI 3-kinases. Although there was no effect on the activity of class Ib PI 3-kinase or on the protein kinase activity of class I PI 3-kinases, there was a biphasic effect on lipid kinase activity of the class Ia enzymes. There was an inhibition of activity at higher peptide concentrations because of a formation of insoluble complexes between peptide and enzyme. Conversely, at lower peptide concentrations there was a profound activation of PI 3-kinase activity of class Ia PI 3-kinases. Studies of peptide variants revealed that all active peptides conform to heptad repeat motifs characteristic of coiled-coil helices. Surface plasmon resonance studies confirmed direct sequence-specific binding of active peptide to the p85alpha adapter subunit of class Ia PI 3-kinase. Active peptides also activated protein kinase B and extracellular signal-regulated kinase (ERK) in vivo in a wortmannin-sensitive manner while reducing recoverable cellular p85 levels. These results indicate that the human lymphocyte antigen class II-derived peptides regulate PI 3-kinase by direct interaction, probably via the coiled-coil domain. These peptides define a novel mechanism of regulating PI 3-kinase and will provide a useful tool for specifically dissecting the function of class Ia PI 3-kinase in cells and for probing structure-function relationships in the class Ia PI 3-kinase heterodimers.

Gene-targeting reveals physiological roles and complex regulation of the phosphoinositide 3-kinases.

Phosphoinositide 3-kinases (PI3Ks) are represented by a family of eight distinct enzymes that can be divided into three classes based on their structure and function. The class I PI3Ks are heterodimeric enzymes that are regulated by recruitment to plasma membrane following receptor activation and which control numerous cellular functions, including growth, differentiation, migration, survival, and metabolism. New light has been shed on the biological role of individual members of the class I PI3Ks and their regulatory subunits through gene-targeting experiments. In addition, these experiments have brought the complexity of how PI3K activation is regulated into focus.

Direct effects of caffeine and theophylline on p110 delta and other phosphoinositide 3-kinases. Differential effects on lipid kinase and protein kinase activities.

We investigated the effects of methylxanthines on enzymatic activity of phosphoinositide 3-kinases (PI3Ks). We found that caffeine inhibits the in vitro lipid kinase of class I PI3Ks (IC(50) = 75 microm for p110 delta, 400 microm for p110 alpha and p110 beta, and 1 mm for p110 gamma), and theophylline has similar effects (IC(50) = 75 microm for p110 delta, 300 microm for p110 alpha, and 800 microm for p110 beta and p110 gamma) and also inhibits the alpha isoform of class II PI3K (PI3K-C2 alpha) (IC(50) approximately 400 microm). However, four other xanthine derivatives tested (3-isobutyl-1-methylxanthine, 3-propylxanthine, alloxazine, and PD116948 (8-cyclopentyl-1,3-dipropylxanthine)) were an order of magnitude less effective. Surprisingly the triazoloquinazoline CGS15943 (9-chloro-2-(2-furyl)(1,2,d)triazolo(1,5-c)quinazolin-5-amine) also selectively inhibits p110 delta (IC(50) < 10 microm). Caffeine and theophylline also inhibit the intrinsic protein kinase activity of the class IA PI3Ks and DNA-dependent protein kinase, although with a much lower potency than that for the lipid kinase (IC(50) approximately 10 mm for p110 alpha, 3 mm for p110 beta, and 10 mm for DNA-dependent protein kinase). In CHO-IR cells and rat soleus muscle, theophylline and caffeine block the ability of insulin to stimulate protein kinase B with IC(50) values similar to those for inhibition of PI3K activity, whereas insulin stimulation of ERK1 or ERK2 was not inhibited at concentrations up to 10 mm. Theophylline and caffeine also blocked insulin stimulation of glucose transport in CHO-IR cells. These results demonstrate that these methylxanthines are direct inhibitors of PI3K lipid kinase activity but are distinctly less effective against serine kinase activity and thus could be of potential use in dissecting these two distinct kinase activities. Theophylline, caffeine, and CGS15943 may be of particular use in dissecting the specific role of the p110 delta lipid kinase. Finally, we conclude that inhibition of PI3K (p110 delta in particular) is likely explain some of the physiological and pharmacological properties of caffeine and theophylline.