PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.

Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P 2 and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyve KO/KO mutant embryos died before the 32-64-cell stage. Cultured fibroblasts derived from PIKfyve flox/flox embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyve WT/KO mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyve WT/KO mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50 -55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P 2 and PtdIns5P selectively decreased, but this reduction (35-40%) was 10 -15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyve WT/KO mice is able to support the demands for PtdIns(3,5)P 2 /PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.Reversible phosphorylation by kinases and phosphatases at positions 3, 4, and/or 5 of the inositol head group in phosphatidylinositol (PtdIns) 2 generates a family of seven phosphoinositide species (1-6). They all are now found to function as versatile membrane-anchored signals that control diverse and essential cellular processes. Consequently, mutations in the genes encoding the phosphoinositide-metabolizing enzymes are associated with an increasing number of human diseases (1-6). The mammalian enzyme that makes PtdIns(3,5)P 2 from PtdIns3P and PtdIns5P from PtdIns is PIKfyve (7,8). It is an evolutionarily conserved large protein of ϳ230 kDa, a product of a single gene in the animal kingdom, whose function is required for proper performance of the endosomal system and certain signaling pathways (9 -11). PIKfyve harbors a PtdIns3P-binding module, i.e. the FYVE finger domain that associates with the PtdIns3P-enriched endosomal membranes, assuring a rapid PIKfyve recruitment to this low abundance substrate of its catalytic activity (12). PIKfyve interacts physically or functionally with multiple partner proteins; the ones involved in PtdIns(3,5)P 2 homeostasis are the best studied (9). Thus, PIKfyve physically associates with the antagonistic enzyme, i.e. the PtdIns(3,5)P 2 -specific phosphatase Sac3, which ...

The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P 2 ) phosphatase Sac3 and ArPIKfyve, the associated regulator of the PtdIns3P-5 kinase PIKfyve, form a stable binary complex that associates with PIKfyve in a ternary complex to increase PtdIns(3,5)P 2 production. I41T rapid loss. Together, our data indentify a novel regulatory mechanism whereby ArPIKfyve enhances Sac3 abundance by attenuating Sac3 proteasome-dependent degradation and suggest that a failure of this mechanism could be the primary molecular defect in the pathogenesis of CMT4J.The phosphatase Sac3 and the kinase PIKfyve 2 are responsible for synthesis and turnover of phosphatidylinositol (PtdIns) (3,5)P 2 in mammalian cells (1). Intriguingly, both the endogenous and the ectopically expressed enzymes are found to reside in the same regulatory complex, called the PAS complex (for PIKfyve-ArPIKfyve-Sac3), organized by the PIKfyve regulator ArPIKfyve and its ability to homooligomerize (2, 3). Despite their antagonistic activities, PIKfyve and Sac3 appear to be enzymatically active in the ternary complex. Thus, in the case of PIKfyve, formation of the PAS regulatory core is critical for PIKfyve activation (3). Likewise, the Sac3 phosphatase retains its PtdIns(3,5)P 2 -hydrolyzing activity within the PAS ternary complex (4). These data reveal an unusual paradigm whereby a common complex relays two opposing activities, one for synthesis, another for degradation, the physiological meaning of which is yet to be understood (1, 5). Data from in vitro reconstitution studies indicate increased and decreased PtdIns(3,5)P 2 levels triggering mammalian endosome fission and fusion, respectively (2, 6). Thus, an association of the two active yet antagonistic enzymes in a common complex would be consistent with the critical requirement for a tight control of PtdIns(3,5)P 2 homeostasis related to dynamic endosome membrane remodeling through fission and fusion (1). Understanding the spatial and temporal regulation and the coordination of the individual enzyme activities within the PAS complex is essential in providing a better comprehension of the intricate PtdIns(3,5)P 2 homeostatic mechanism. Maintaining PtdIns(3,5)P 2 homeostasis is apparently indispensable for life as evidenced by the early lethality of Drosophila melanogaster or Caenorhabditis elegans PIKfyve-null mutants (7,8) and by the early death of ArPIKfyve and Sac3-deficient mouse models (9, 10). Concordantly, a defective Sac3 I41T allele in combination with a null allele is responsible for the pathogenesis of Charcot-Marie-Tooth type 4J (CMT4J) peripheral neuropathy, a recessively inherited disease with early onset manifested by progressive motor and sensory neuron degeneration (10, 11). The molecular and cellular mechanisms rendering this single I41T amino acid substitution pathogenic are currently unknown.ArPIKfyve associates with the Sac3 phosphatase independently of PIKfyve in a stable ArPIKfyve-Sac3 heterooligomer (3,4). This binary association is apparently a prerequisite for a produc...

The evolutionarily conserved kinase PIKfyve that synthesizes PtdIns5P and PtdIns(3,5)P₂ has been implicated in insulin-regulated GLUT4 translocation/glucose entry in 3T3-L1 adipocytes. To decipher PIKfyve's role in muscle and systemic glucose metabolism, here we have developed a novel mouse model with Pikfyve gene disruption in striated muscle (MPIfKO). These mice exhibited systemic glucose intolerance and insulin resistance at an early age but had unaltered muscle mass or proportion of slow/fast-twitch muscle fibers. Insulin stimulation of in vivo or ex vivo glucose uptake and GLUT4 surface translocation was severely blunted in skeletal muscle. These changes were associated with premature attenuation of Akt phosphorylation in response to in vivo insulin, as tested in young mice. Starting at 10-11 wk of age, MPIfKO mice progressively accumulated greater body weight and fat mass. Despite increased adiposity, serum free fatty acid and triglyceride levels were normal until adulthood. Together with the undetectable lipid accumulation in liver, these data suggest that lipotoxicity and muscle fiber switching do not contribute to muscle insulin resistance in MPIfKO mice. Furthermore, the 80% increase in total fat mass resulted from increased fat cell size rather than altered fat cell number. The observed profound hyperinsulinemia combined with the documented increases in constitutive Akt activation, in vivo glucose uptake, and gene expression of key enzymes for fatty acid biosynthesis in MPIfKO fat tissue suggest that the latter is being sensitized for de novo lipid anabolism. Our data provide the first in vivo evidence that PIKfyve is essential for systemic glucose homeostasis and insulin-regulated glucose uptake/GLUT4 translocation in skeletal muscle.

Summary While adverse effects of prolonged recipient warm ischemia time (rWIT) in liver transplantation (LT) have been well investigated, few studies have focused on possible positive prognostic effects of short rWIT. We aim to investigate if shortening rWIT can further improve outcomes in donation after brain death liver transplant (DBD‐LT). Primary DBD‐LT between 2000 and 2019 were retrospectively reviewed. Patients were divided according to rWIT (≤30, 31–40, 41–50, and >50 min). The requirement of intraoperative transfusion, early allograft dysfunction (EAD), and graft survival were compared between the rWIT groups. A total of 1,256 patients of DBD‐LTs were eligible. rWIT was ≤30min in 203 patients (15.7%), 31–40min in 465 patients (37.3%), 41–50min in 353 patients (28.1%), and >50min in 240 patients (19.1%). There were significant increasing trends of transfusion requirement (P < 0.001) and increased estimated blood loss (EBL, P < 0.001), and higher lactate level (P < 0.001) with prolongation of rWIT. Multivariable logistic regression demonstrated the lowest risk of EAD in the WIT ≤30min group. After risk adjustment, patients with rWIT ≤30 min showed a significantly lower risk of graft loss at 1 and 5‐years, compared to other groups. The positive prognostic impact of rWIT ≤30min was more prominent when cold ischemia time exceeded 6 h. In conclusion, shorter rWIT in DBD‐LT provided significantly better post‐transplant outcomes.

Neurofibromatosis type 1 is a congenital condition affecting neurons and connective tissue integrity including vasculature. On extremely rare occasions these patients present with venous aneurysms affecting the internal jugular vein. If they become large enough there presents a risk of rupture, thrombosis, embolization or compression of adjacent structures. In these circumstances, or when the patient becomes symptomatic, surgical exploration is warranted. We present a case of one of the largest aneurysms in the literature and one of only five associated with Neurofibromatosis type 1. A 63-year-old female who initially presented for a Hinchey III diverticulitis requiring laparotomy developed an incidentally discovered left neck swelling prior to discharge. After nonspecific clinical exam findings, imaging identified a thrombosed internal jugular vein aneurysm. Due to the risks associated with the particularly large size of our patient’s aneurysm, our patient underwent surgical exploration with ligation and excision. Although several techniques have been reported, for similar presentations, we recommend this technique.

Systemic deficiency of PIKfyve, the evolutionarily conserved phosphoinositide kinase synthesizing cellular PtdIns5P and PtdIns(3,5)P2 and implicated in insulin signaling, causes early embryonic death in mice. In contrast, mice with muscle‐specific Pikfyve disruption have normal lifespan but exhibit early‐age whole‐body glucose intolerance and muscle insulin resistance, thus establishing the key role of muscle PIKfyve in glucose homeostasis. Fat and muscle tissues control postprandial glucose clearance through different mechanisms, raising questions as to whether adipose Pikfyve disruption will also trigger whole‐body metabolic abnormalities, and if so, what the mechanism might be. To clarify these issues, here we have characterized two new mouse models with adipose tissue disruption of Pikfyve through Cre recombinase expression driven by adipose‐specific aP2‐ or adiponectin (Aq) promoters. Whereas both mouse lines were ostensibly normal until adulthood, their glucose homeostasis and systemic insulin sensitivity were severely dysregulated. These abnormalities stemmed in part from accelerated fat‐cell lipolysis and elevated serum FFA. Intriguingly, aP2‐Cre‐PIKfyvefl/fl but not Aq‐Cre‐PIKfyvefl/fl females had severely impaired pregnancy‐induced mammary gland differentiation and lactogenesis, consistent with aP2‐Cre‐mediated Pikfyve excision in nonadipogenic tissues underlying this defect. Intriguingly, whereas mammary glands from postpartum control and Aq‐Cre‐PIKfyvefl/fl mice or ex vivo mammary gland explants showed profound upregulation of PIKfyve protein levels subsequent to prolactin receptor activation, such increases were not apparent in aP2‐Cre‐PIKfyvefl/fl females. Collectively, our data identify for the first time that adipose tissue Pikfyve plays a key role in the mechanisms regulating glucose homeostasis and that the PIKfyve pathway is critical in mammary epithelial differentiation during pregnancy and lactogenesis downstream of prolactin receptor signaling.

No abstract