PI3K isoforms in cell signalling and vesicle trafficking.

PI3Ks are a family of lipid kinases that phosphorylate intracellular inositol lipids to regulate signalling and intracellular vesicular traffic. Mammals have eight isoforms of PI3K, divided into three classes. The class I PI3Ks generate 3-phosphoinositide lipids, which directly activate signal transduction pathways. In addition to being frequently genetically activated in cancer, similar mutations in class I PI3Ks have now also been found in a human non-malignant overgrowth syndrome and a primary immune disorder that predisposes to lymphoma. The class II and class III PI3Ks are regulators of membrane traffic along the endocytic route, in endosomal recycling and autophagy, with an often indirect effect on cell signalling. Here, we summarize current knowledge of the different PI3K classes and isoforms, focusing on recently uncovered biological functions and the mechanisms by which these kinases are activated. Deeper insight into the PI3K isoforms will undoubtedly continue to contribute to a better understanding of fundamental cell biological processes and, ultimately, of human disease.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

The emerging mechanisms of isoform-specific PI3K signalling.

Phosphoinositide 3-kinases (PI3Ks) function early in intracellular signal transduction pathways and affect many biological functions. A further level of complexity derives from the existence of eight PI3K isoforms, which are divided into class I, class II and class III PI3Ks. PI3K signalling has been implicated in metabolic control, immunity, angiogenesis and cardiovascular homeostasis, and is one of the most frequently deregulated pathways in cancer. PI3K inhibitors have recently entered clinical trials in oncology. A better understanding of how the different PI3K isoforms are regulated and control signalling could uncover their roles in pathology and reveal in which disease contexts their blockade could be most beneficial.

Uninephrectomy and class II PI3K-C2ß inactivation synergistically protect against obesity, insulin resistance and liver steatosis in mice.

Uninephrectomy (UNx) in living kidney donors for transplantation is now routine clinical practice. While chronic kidney disease, due to bilateral kidney dysfunction, is associated with insulin resistance, liver steatosis, and type 2 diabetes, the metabolic impact of UNx remains unclear. To better understand the crosstalk between the kidney and insulin target tissues, we studied the metabolic consequences of UNx and the potential involvement of class II PI3K-C2ß, the inactivation of which has been reported to result in insulin sensitization. Mice underwent UNx or sham operation followed by either normal chow or high-fat diet (HFD). Seventeen weeks post-UNx, mice showed improved glucose tolerance, insulin sensitivity, and decreased HFD-induced liver steatosis. This was associated with an enhanced serum FGF21 and insulin-stimulated Akt signaling in the liver and muscle of both lean and obese mice. Remarkably, the combination of UNx and PI3K-C2ß inactivation protected against HFD-induced obesity and further potentiated the metabolic improvement observed in WT UNx mice correlating with a synergistic increase in metabolic tissues of (1) insulin-stimulated Akt signaling (2) FGFR1 and ßKlotho expression. We demonstrated a potential beneficial effect of kidney donation and more effectively with PI3K-C2ß inactivation to protect against metabolic disorders through a mutual insulin/FGF21 sensitization.

A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth.

To uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.

Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition.

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.

Environmental stress affects the activity of metabolic and growth factor signaling networks and induces autophagy markers in MCF7 breast cancer cells.

Phosphoproteomic techniques are contributing to our understanding of how signaling pathways interact and regulate biological processes. This technology is also being used to characterize how signaling networks are remodeled during disease progression and to identify biomarkers of signaling pathway activity and of responses to cancer therapy. A potential caveat in these studies is that phosphorylation is a very dynamic modification that can substantially change during the course of an experiment or the retrieval and processing of cellular samples. Here, we investigated how exposure of cells to ambient conditions modulates phosphorylation and signaling pathway activity in the MCF7 breast cancer cell line. About 1.5% of 3,500 sites measured showed a significant change in phosphorylation extent upon exposure of cells to ambient conditions for 15 min. The effects of this perturbation in modifying phosphorylation patterns did not involve random changes due to stochastic activation of kinases and phosphatases. Instead, exposure of cells to ambient conditions elicited an environmental stress reaction that involved a coordinated response to a metabolic stress situation, which included: (1) the activation of AMPK; (2) the inhibition of PI3K, AKT, and ERK; (3) an increase in markers of protein synthesis inhibition at the level of translation elongation; and (4) an increase in autophagy markers. We also observed that maintaining cells in ice modified but did not completely abolish this metabolic stress response. In summary, exposure of cells to ambient conditions affects the activity of signaling networks previously implicated in metabolic and growth factor signaling. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000472.

Class III phosphoinositide 3-kinase/VPS34 and dynamin are critical for apical endocytic recycling.

Recycling is a limiting step for receptor-mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan-class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP-2, causing surface down-regulation. GFP-(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002-treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin-GFP and dependence of dynamin-GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K-III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.

Cinderella finds her shoe: the first Vps34 inhibitor uncovers a new PI3K-AGC protein kinase connection.

Class II/III PI3Ks (phosphoinositide 3-kinases) produce the PtdIns(3)P lipid that is involved in intracellular vesicular trafficking. In contrast with class I PI3Ks, the potential signalling roles of class II/III PI3Ks are poorly understood. In a recent article in the Biochemical Journal, Bago and co-workers report that Vps34 (vacuolar protein sorting 34), the only class III PI3K, controls the activity of SGK3 (serum- and glucocorticoid-regulated protein kinase 3). Like other AGC kinases, the SGKs (SGK1, SGK2 and SGK3) are activated by dual phosphorylation. Unlike its cousins SGK1 and SGK2, SGK3 contains a PtdIns(3)P-binding domain, providing an additional element of regulation. The study by Bago et al. characterizes and makes extensive use of a Novartis Vps34 inhibitor (VPS34-IN1) that inhibits this PI3K isoform with nanomolar potency, without affecting other lipid kinases or more than 300 protein kinases. The authors show that this compound very rapidly reduced PtdIns(3)P levels at the endosome with concomitant loss of SGK3 phosphorylation. Co-inhibition of class I PI3Ks led to a further reduction in SGK3 activity, indicating that class I PI3Ks may also regulate SGK3 activity through an additional, currently unknown, mechanism. It remains to be assessed whether the novel PI3K-protein kinase connection established by this study is subject to acute cellular stimulation or is part of a constitutive housekeeping function. VPS34-IN1 will provide a useful tool to decipher the kinase-dependent functions of Vps34, with acute changes in SGK3 phosphorylation and subcellular localization being new biomarkers of Vps34 activity.

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover.

Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2ß, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2ß. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.

A new tool to dissect the function of p70 S6 kinase.

Developing small-molecule inhibitors that are highly selective for specific protein kinases has been and remains a serious challenge. This especially applies to members of families of related kinases with overlapping substrate specificities, such as the serine/threonine kinases of the AGC family. In this issue of the Biochemical Journal, Dario Alessi's group, in a collaboration with Pfizer, report on PF-4708671, a potent and highly selective inhibitor of S6K1 (p70 S6 kinase 1) in vitro and in cells. S6K1 is an AGC family member and a crucial effector of the mTORC1 (mammalian target of rapamycin complex 1) kinase. This is the first reported inhibitor that is highly selective for S6K1. This compound will help to understand the signalling and physiological roles of S6K1, and to dissect signalling downstream of mTORC1. S6K1 inhibitors may ultimately be useful in the treatment of diseases such as cancer where S6K1 is overexpressed, but most importantly in metabolic disease such as insulin resistance and obesity.

Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation.

Mutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy - tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient-derived cells carrying the most prevalent disease related mtDNA mutation, the m.3243 A > G. These studies reveal that the mutation promotes changes in metabolites which are associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 reduced mtDNA mutant load and partially rescued cellular bioenergetic function. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243 A > G mutation.

Tuberous sclerosis complex proteins 1 and 2 control serum-dependent translation in a TOP-dependent and -independent manner.

The tuberous sclerosis complex (TSC) proteins TSC1 and TSC2 regulate protein translation by inhibiting the serine/threonine kinase mTORC1 (for mammalian target of rapamycin complex 1). However, how TSC1 and TSC2 control overall protein synthesis and the translation of specific mRNAs in response to different mitogenic and nutritional stimuli is largely unknown. We show here that serum withdrawal inhibits mTORC1 signaling, causes disassembly of translation initiation complexes, and causes mRNA redistribution from polysomes to subpolysomes in wild-type mouse embryo fibroblasts (MEFs). In contrast, these responses are defective in Tsc1(-/-) or Tsc2(-/-) MEFs. Microarray analysis of polysome- and subpolysome-associated mRNAs uncovered specific mRNAs that are translationally regulated by serum, 90% of which are TSC1 and TSC2 dependent. Surprisingly, the mTORC1 inhibitor, rapamycin, abolished mTORC1 activity but only affected approximately 40% of the serum-regulated mRNAs. Serum-dependent signaling through mTORC1 and polysome redistribution of global and individual mRNAs were restored upon re-expression of TSC1 and TSC2. Serum-responsive mRNAs that are sensitive to inhibition by rapamycin are highly enriched for terminal oligopyrimidine and for very short 5' and 3' untranslated regions. These data demonstrate that the TSC1/TSC2 complex regulates protein translation through mainly mTORC1-dependent mechanisms and implicates a discrete profile of deregulated mRNA translation in tuberous sclerosis pathology.

Class III PI3K Vps34 Controls Thyroid Hormone Production by Regulating Thyroglobulin Iodination, Lysosomal Proteolysis, and Tissue Homeostasis.

Background: The production of thyroid hormones [triiodothyronine (T3), thyroxine (T4)] depends on the organization of the thyroid in follicles, which are lined by a monolayer of thyrocytes with strict apicobasal polarity. This polarization supports vectorial transport of thyroglobulin (Tg) for storage into, and recapture from, the colloid. It also allows selective addressing of channels, transporters, ion pumps, and enzymes to their appropriate basolateral [Na+/I- symporter (NIS), SLC26A7, and Na+/K+-ATPase] or apical membrane domain (anoctamin, SLC26A4, DUOX2, DUOXA2, and thyroperoxidase). How these actors of T3/T4 synthesis reach their final destination remains poorly understood. The PI 3-kinase isoform Vps34/PIK3C3 is now recognized as a main component in the general control of vesicular trafficking and of cell homeostasis through the regulation of endosomal trafficking and autophagy. We recently reported that conditional Vps34 inactivation in proximal tubular cells in the kidney prevents normal addressing of apical membrane proteins and causes abortive macroautophagy. Methods:Vps34 was inactivated using a Pax8-driven Cre recombinase system. The impact of Vps34 inactivation in thyrocytes was analyzed by histological, immunolocalization, and messenger RNA expression profiling. Thyroid hormone synthesis was assayed by 125I injection and plasma analysis. Results:Vps34 conditional knockout (Vps34cKO) mice were born at the expected Mendelian ratio and showed normal growth until postnatal day 14 (P14), then stopped growing and died at ∼1 month of age. We therefore analyzed thyroid Vps34cKO at P14. We found that loss of Vps34 in thyrocytes causes (i) disorganization of thyroid parenchyma, with abnormal thyrocyte and follicular shape and reduced PAS+ colloidal spaces; (ii) severe noncompensated hypothyroidism with extremely low T4 levels (0.75 ± 0.62 µg/dL) and huge thyrotropin plasma levels (19,300 ± 10,500 mU/L); (iii) impaired 125I organification at comparable uptake and frequent occurrence of follicles with luminal Tg but nondetectable T4-bearing Tg; (iv) intense signal in thyrocytes for the lysosomal membrane marker, LAMP-1, as well as Tg and the autophagy marker, p62, indicating defective lysosomal proteolysis; and (v) presence of macrophages in the colloidal space. Conclusions: We conclude that Vps34 is crucial for thyroid hormonogenesis, at least by controlling epithelial organization, Tg iodination as well as proteolytic T3/T4 excision in lysosomes.

Perspective: Potential Impact and Therapeutic Implications of Oncogenic PI3K Activation on Chromosomal Instability.

Genetic activation of the class I PI3K pathway is very common in cancer. This mostly results from oncogenic mutations in PIK3CA, the gene encoding the ubiquitously expressed PI3Kα catalytic subunit, or from inactivation of the PTEN tumour suppressor, a lipid phosphatase that opposes class I PI3K signalling. The clinical impact of PI3K inhibitors in solid tumours, aimed at dampening cancer-cell-intrinsic PI3K activity, has thus far been limited. Challenges include poor drug tolerance, incomplete pathway inhibition and pre-existing or inhibitor-induced resistance. The principle of pharmacologically targeting cancer-cell-intrinsic PI3K activity also assumes that all cancer-promoting effects of PI3K activation are reversible, which might not be the case. Emerging evidence suggests that genetic PI3K pathway activation can induce and/or allow cells to tolerate chromosomal instability, which-even if occurring in a low fraction of the cell population-might help to facilitate and/or drive tumour evolution. While it is clear that such genomic events cannot be reverted pharmacologically, a role for PI3K in the regulation of chromosomal instability could be exploited by using PI3K pathway inhibitors to prevent those genomic events from happening and/or reduce the pace at which they are occurring, thereby dampening cancer development or progression. Such an impact might be most effective in tumours with clonal PI3K activation and achievable at lower drug doses than the maximum-tolerated doses of PI3K inhibitors currently used in the clinic.

Translational deregulation in PDK-1-/- embryonic stem cells.

PDK-1 is a protein kinase that is critical for the activation of many downstream protein kinases in the AGC superfamily, through phosphorylation of the activation loop site on these substrates. Cells lacking PDK-1 show decreased activity of these protein kinases, including protein kinase B (PKB) and p70S6K, whereas mTOR activity remains largely unaffected. Here we show, by assessing both association of cellular RNAs with polysomes and by metabolic labeling, that PDK-1-/- embryonic stem (ES) cells exhibit defects in mRNA translation. We identify which mRNAs are most dramatically translationally regulated in cells lacking PDK-1 expression by performing microarray analysis of total and polysomal RNA in these cells. In addition to the decreased translation of many RNAs, a smaller number of RNAs show increased association with polyribosomes in PDK-1-/- ES cells relative to PDK-1+/+ ES cells. We show that PKB activity is a critical downstream component of PDK-1 in mediating translation of cystatin C, RANKL, and Rab11a, whereas mTOR activity is less important for effective translation of these targets.

Vps34/PI3KC3 deletion in kidney proximal tubules impairs apical trafficking and blocks autophagic flux, causing a Fanconi-like syndrome and renal insufficiency.

Kidney proximal tubular cells (PTCs) are highly specialized for ultrafiltrate reabsorption and serve as paradigm of apical epithelial differentiation. Vps34/PI3-kinase type III (PI3KC3) regulates endosomal dynamics, macroautophagy and lysosomal function. However, its in vivo role in PTCs has not been evaluated. Conditional deletion of Vps34/PI3KC3 in PTCs by Pax8-Cre resulted in early (P7) PTC dysfunction, manifested by Fanconi-like syndrome, followed by kidney failure (P14) and death. By confocal microscopy, Vps34∆/∆ PTCs showed preserved apico-basal specification (brush border, NHERF-1 versus Na+/K+-ATPase, ankyrin-G) but basal redistribution of late-endosomes/lysosomes (LAMP-1) and mis-localization to lysosomes of apical recycling endocytic receptors (megalin, cubilin) and apical non-recycling solute carriers (NaPi-IIa, SGLT-2). Defective endocytosis was confirmed by Texas-red-ovalbumin tracing and reduced albumin content. Disruption of Rab-11 and perinuclear galectin-3 compartments suggested mechanistic clues for defective receptor recycling and apical biosynthetic trafficking. p62-dependent autophagy was triggered yet abortive (p62 co-localization with LC3 but not LAMP-1) and PTCs became vacuolated. Impaired lysosomal positioning and blocked autophagy are known causes of cell stress. Thus, early trafficking defects show that Vps34 is a key in vivo component of molecular machineries governing apical vesicular trafficking, thus absorptive function in PTCs. Functional defects underline the essential role of Vps34 for PTC homeostasis and kidney survival.

Vps34 PI 3-kinase inactivation enhances insulin sensitivity through reprogramming of mitochondrial metabolism.

Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.

Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi.

RNAi (RNA interference) and ASO (antisense oligonucleotide) technologies are the most commonly used approaches for silencing gene expression. However, the specificity of such powerful tools is an important factor to correctly interpret the biological consequences of gene silencing. In the present study, we examined the effects of acute loss of Ser/Thr kinase PDK1 (3-phosphoinositide-dependent kinase 1) expression using ASO and RNAi, and compared, for the first time, these two techniques using Affymetrix microarrays. We show that both ASO- and siRNA (small interfering RNA)-mediated knock-down of PDK1 expression strongly inhibited cell proliferation, although by different mechanisms, thereby questioning the specificity of these reagents. Using microarray analysis, we characterized the specificity of the ASO- and siRNA-mediated gene silencing of PDK1 by examining expression profiles 48 and 72 h following oligonucleotide transfection. At 48 h, a PDK1-dependent pattern of gene alterations was detectable, despite a large number of non-specific changes due to transfection of control nucleic acids. These non-specific alterations became more apparent at the 72 h time point, and obscured any PDK1-specific pattern. This study underscores the importance of defining appropriate control ASOs and siRNAs, using multiple oligonucleotides for each target and preferably short time points following transfection to avoid misinterpretation of the phenotype observed.