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van Horck, Francis P. G., Emmanuelle Lavazais, Britta J. Eickholt, Wouter H. Moolenaar, and Nullin Divecha. "Essential Role of Type Iα Phosphatidylinositol 4-Phosphate 5-Kinase in Neurite Remodeling." Current Biology 12, no. 3 (February 2002): 241–45. http://dx.doi.org/10.1016/s0960-9822(01)00660-1.
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Shim, Hyeseok, Chuan Wu, Shivan Ramsamooj, Kaitlyn N. Bosch, Zuojia Chen, Brooke M. Emerling, Jihye Yun, et al. "Deletion of the gene Pip4k2c, a novel phosphatidylinositol kinase, results in hyperactivation of the immune system." Proceedings of the National Academy of Sciences 113, no. 27 (June 16, 2016): 7596–601. http://dx.doi.org/10.1073/pnas.1600934113.
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Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three enzymes PI5P4Kα, PI5P4Kβ, and PI5P4Kγ, and these enzymes have been implicated in metabolic control, growth control, and a variety of stress responses. Here, we show that mice with germline deletion of type 2 phosphatidylinositol-5-phosphate 4-kinase gamma (Pip4k2c), the gene encoding PI5P4Kγ, appear normal in regard to growth and viability but have increased inflammation and T-cell activation as they age. Immune cell infiltrates increased in Pip4k2c−/− mouse tissues. Also, there was an increase in proinflammatory cytokines, including IFNγ, interleukin 12, and interleukin 2 in plasma of Pip4k2c−/− mice. Pip4k2c−/− mice had an increase in T-helper-cell populations and a decrease in regulatory T-cell populations with increased proliferation of T cells. Interestingly, mammalian target of rapamycin complex 1 (mTORC1) signaling was hyperactivated in several tissues from Pip4k2c−/− mice and treating Pip4k2c−/− mice with rapamycin reduced the inflammatory phenotype, resulting in a decrease in mTORC1 signaling in tissues and a decrease in proinflammatory cytokines in plasma. These results indicate that PI5P4Kγ plays a role in the regulation of the immune system via mTORC1 signaling.
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Bridges, Dave, Jing-Tyan Ma, Sujin Park, Ken Inoki, Lois S. Weisman, and Alan R. Saltiel. "Phosphatidylinositol 3,5-bisphosphate plays a role in the activation and subcellular localization of mechanistic target of rapamycin 1." Molecular Biology of the Cell 23, no. 15 (August 2012): 2955–62. http://dx.doi.org/10.1091/mbc.e11-12-1034.
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The kinase complex mechanistic target of rapamycin 1 (mTORC1) plays an important role in controlling growth and metabolism. We report here that the stepwise formation of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) regulates the cell type–specific activation and localization of mTORC1. PI(3)P formation depends on the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α, as well as the class III PI3K Vps34, while PI(3,5)P2 requires the phosphatidylinositol-3-phosphate-5-kinase PIKFYVE. In this paper, we show that PIKFYVE and PI3K-C2α are necessary for activation of mTORC1 and its translocation to the plasma membrane in 3T3-L1 adipocytes. Furthermore, the mTORC1 component Raptor directly interacts with PI(3,5)P2. Together these results suggest that PI(3,5)P2 is an essential mTORC1 regulator that defines the localization of the complex.
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Prasad, K. V., R. Kapeller, O. Janssen, H. Repke, J. S. Duke-Cohan, L. C. Cantley, and C. E. Rudd. "Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase." Molecular and Cellular Biology 13, no. 12 (December 1993): 7708–17. http://dx.doi.org/10.1128/mcb.13.12.7708-7717.1993.
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CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.
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Prasad, K. V., R. Kapeller, O. Janssen, H. Repke, J. S. Duke-Cohan, L. C. Cantley, and C. E. Rudd. "Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase." Molecular and Cellular Biology 13, no. 12 (December 1993): 7708–17. http://dx.doi.org/10.1128/mcb.13.12.7708.
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CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.
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Davis, J. N., C. O. Rock, M. Cheng, J. B. Watson, R. A. Ashmun, H. Kirk, R. J. Kay, and M. F. Roussel. "Complementation of growth factor receptor-dependent mitogenic signaling by a truncated type I phosphatidylinositol 4-phosphate 5-kinase." Molecular and Cellular Biology 17, no. 12 (December 1997): 7398–406. http://dx.doi.org/10.1128/mcb.17.12.7398.
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Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When we transfected cells expressing CSF-1R (Y809F) with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 was restored and ligand-dependent cell proliferation was sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilized CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensated for a mitogenically defective CSF-1R by interfering with this process.
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Demian, Douglas J., Susan L. Clugston, Meta M. Foster, Lucia Rameh, Deborah Sarkes, Sharon A. Townson, Lily Yang, Melvin Zhang, and Maura E. Charlton. "High-Throughput, Cell-Free, Liposome-Based Approach for Assessing In Vitro Activity of Lipid Kinases." Journal of Biomolecular Screening 14, no. 7 (July 29, 2009): 838–44. http://dx.doi.org/10.1177/1087057109339205.
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Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [γ-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIβ and α) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIβ phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets. ( Journal of Biomolecular Screening 2009:838-844)
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Zhang, Jiping, Ruihua Luo, Heqing Wu, Shunhui Wei, Weiping Han, and GuoDong Li. "Role of Type Iα Phosphatidylinositol-4-Phosphate 5-Kinase in Insulin Secretion, Glucose Metabolism, and Membrane Potential in INS-1 β-Cells." Endocrinology 150, no. 5 (December 30, 2008): 2127–35. http://dx.doi.org/10.1210/en.2008-0516.
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Insulin secretion from β-cells is regulated by a complex signaling network. Our earlier study has reported that Rac1 participates in glucose- and cAMP-induced insulin secretion probably via maintaining a functional actin structure for recruitment of insulin granules. Type Iα phosphatidylinositol-4-phosphate 5-kinase (PIP5K-Iα) is a downstream effector of Rac1 and a critical enzyme for synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2). By using an RNA interference technique, PIP5K-Iα in INS-1 β-cells could be specifically knocked down by 70–75%. PIP5K-Iα knockdown disrupted filamentous actin structure and caused changes in cell morphology. In addition, PIP2 content in the plasma membrane was reduced and the glucose effect on PIP2 was abolished but without affecting glucose-induced formation of inositol 1,4,5-trisphosphate. At basal conditions (2.8 mm glucose), PIP5K-Iα knockdown doubled insulin secretion, elevated glucose metabolic rate, depolarized resting membrane potential, and raised cytoplasmic free Ca2+ levels ([Ca2+]i). The total insulin release at high glucose was increased upon PIP5K-Iα knockdown. However, the percent increment of insulin secretion by high glucose and forskolin over the basal release was significantly reduced, an effect more apparent on the late phase of insulin secretion. Metabolism and [Ca2+]i rises at high glucose were also attenuated in cells after PIP5K-Iα knockdown. In contrast, PIP5K-Iα knockdown had no effect on cell growth and viability. Taken together, our data suggest that PIP5K-Iα may play an important role in both the proximal and distal steps of signaling cascade for insulin secretion in β-cells.
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Jones, David H., James B. Morris, Clive P. Morgan, Hisatake Kondo, Robin F. Irvine, and Shamshad Cockcroft. "Type I Phosphatidylinositol 4-Phosphate 5-Kinase Directly Interacts with ADP-ribosylation Factor 1 and Is Responsible for Phosphatidylinositol 4,5-Bisphosphate Synthesis in the Golgi Compartment." Journal of Biological Chemistry 275, no. 18 (March 9, 2000): 13962–66. http://dx.doi.org/10.1074/jbc.c901019199.
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Gerber, Pehuén Pereyra, Mercedes Cabrini, Carolina Jancic, Luciana Paoletti, Claudia Banchio, Catalina von Bilderling, Lorena Sigaut, et al. "Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate." Journal of Cell Biology 209, no. 3 (May 4, 2015): 435–52. http://dx.doi.org/10.1083/jcb.201409082.
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During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.
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Yamamoto, Masaya, Mark Z. Chen, Ying-Jie Wang, Hui-Qiao Sun, Yongjie Wei, Manuel Martinez, and Helen L. Yin. "Hypertonic Stress Increases Phosphatidylinositol 4,5-Bisphosphate Levels by Activating PIP5KIβ." Journal of Biological Chemistry 281, no. 43 (August 30, 2006): 32630–38. http://dx.doi.org/10.1074/jbc.m605928200.
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Hyperosmotic stress increases phosphoinositide levels, reorganizes the actin cytoskeleton, and induces multiple acute and adaptive physiological responses. Here we showed that phosphatidylinositol 4,5-bisphosphate (PIP2) level increased rapidly in HeLa cells during hypertonic treatment. Depletion of the human type I phosphatidylinositol 4-phosphate 5-kinase β isoform (PIP5KIβ) by RNA interference impaired both the PIP2 and actin cytoskeletal responses. PIP5KIβ was recruited to membranes and was activated by hypertonic stress through Ser/Thr dephosphorylation. Calyculin A, a protein phosphatase 1 inhibitor, blocked the hypertonicity-induced PIP5KIβ dephosphorylation/activation as well as PIP2 increase in cells. Urea, which raises osmolarity without inducing cell shrinkage, did not promote dephosphorylation nor increase PIP2 levels. Disruption or stabilization of the actin cytoskeleton, or inhibition of the Rho kinase, did not block the PIP2 increase nor PIP5KIβ dephosphorylation. Therefore, PIP5KIβ is dephosphorylated in a volume-dependent manner by a calyculin A-sensitive protein phosphatase, which is activated upstream of actin remodeling and independently of Rho kinase activation. Our results establish a cause-and-effect relation between PIP5KIβ dephosphorylation, lipid kinase activation, and PIP2 increase in cells. This PIP2 increase can orchestrate multiple downstream responses, including the reorganization of the actin cytoskeleton.
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Yan, Qinnan, Huanqing Gao, Qing Yao, Kun Ling, and Guozhi Xiao. "Loss of phosphatidylinositol-4-phosphate 5-kinase type-1 gamma (Pip5k1c) in mesenchymal stem cells leads to osteopenia by impairing bone remodeling." Journal of Biological Chemistry 298, no. 3 (March 2022): 101639. http://dx.doi.org/10.1016/j.jbc.2022.101639.
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PANARETOU, Christina, and Sharon A. TOOZE. "Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation factor 1." Biochemical Journal 363, no. 2 (April 8, 2002): 289–95. http://dx.doi.org/10.1042/bj3630289.
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Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive Gi/G0 proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913–16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on Gi/G0 activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1—GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280–287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962–13170], whereas ARF1—GTP does regulate the production of PtdIns(4,5)P2. Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.
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Kunii, Yasuto, Junya Matsumoto, Ryuta Izumi, Atsuko Nagaoka, Mizuki Hino, Risa Shishido, Makoto Sainouchi, et al. "Evidence for Altered Phosphoinositide Signaling-Associated Molecules in the Postmortem Prefrontal Cortex of Patients with Schizophrenia." International Journal of Molecular Sciences 22, no. 15 (July 31, 2021): 8280. http://dx.doi.org/10.3390/ijms22158280.
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Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3β (GSK3β) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3β expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.
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Xie, Y., L. Zhu, and G. Zhao. "Assignment1 of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5K1A) to human chromosome bands 1q22→ q24 by in situ hybridization." Cytogenetic and Genome Research 88, no. 3-4 (2000): 197–99. http://dx.doi.org/10.1159/000015545.
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Mikhalitskaya, E. V., O. V. Roshchina, S. A. Ivanova, and N. A. Bokhan. "Study of the polymorphic variants of the PIP5K2A gene association with the comorbidity of alcoholism and affective disorders." V.M. BEKHTEREV REVIEW OF PSYCHIATRY AND MEDICAL PSYCHOLOGY, no. 4-1 (December 9, 2019): 124–26. http://dx.doi.org/10.31363/2313-7053-2019-4-1-124-126.
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One of the common pathogenetic mechanisms of the formation of alcohol dependence and depressive disorders can be a violation of the neurotransmitter systems, in particular — dopamine. Phosphatidylinositol-4-phosphate-5-kinase type 2 alpha (PIP5K2A) plays an important role in the regulation of neuronal excitability and synaptic dopamine neurotransmission. The aim of this study was to assess the presence of associations of the PIP5K2A gene polymorphic variants with the comorbid course of alcohol dependence and depressive disorders. This study showed differences in the frequency of the genotype distribution of 3 PIP5K2A gene polymorphisms (rs946961, rs1132816, and rs1417374) both between patient groups compared with the control group, and between the patient group and the group with the comorbid course of disorders.
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Mace, Emily M., Jinyi Zhang, Katherine A. Siminovitch, and Fumio Takei. "Elucidation of the integrin LFA-1–mediated signaling pathway of actin polarization in natural killer cells." Blood 116, no. 8 (August 26, 2010): 1272–79. http://dx.doi.org/10.1182/blood-2009-12-261487.
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Abstract The leukocyte integrin LFA-1 is critical for natural killer (NK) cell cytotoxicity as it mediates NK-cell adhesion to target cells and generates activating signals that lead to polarization of the actin cytoskeleton. However, the LFA-1–mediated signaling pathway is not fully understood. Here, we examined the subcellular localization of actin-associated proteins in wild-type, talin-deficient, and Wiskott-Aldrich Syndrome protein (WASP)–deficient NK cells bound to beads coated with the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). In addition, we carried out coimmunoprecipitation analyses and also used a pharmacologic reagent to reduce the level of phosphatidylinositol-4,5-bisphosphate (PIP2). The results revealed the following signaling pathways. Upon ICAM-1 binding to LFA-1, talin redistributes to the site of LFA-1 ligation and initiates 2 signaling pathways. First, talin recruits the actin nucleating protein complex Arp2/3 via constitutive association of vinculin with talin and Arp2/3. Second, talin also associates with type I phosphatidylinositol 4-phosphate 5-kinase (PIPKI) and binding of LFA-1 to ICAM-1 results in localized increase in PIP2. This increase in PIP2 recruits WASP to the site of LFA-1 ligation where WASP promotes Arp2/3-mediated actin polymerization. These processes are critical for the initiation of NK cell–mediated cytotoxicity.
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Kawase, Atsushi, Yuta Inoue, Miho Hirosoko, Yuka Sugihara, Hiroaki Shimada, and Masahiro Iwaki. "Decrease in Multidrug Resistance-associated Protein 2 Activities by Knockdown of Phosphatidylinositol 4-phosphate 5-kinase in Hepatocytes and Cancer Cells." Journal of Pharmacy & Pharmaceutical Sciences 22 (November 19, 2019): 576–84. http://dx.doi.org/10.18433/jpps30444.
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Purpose: The plasma membrane localization and transport activity of multidrug resistance-associated protein 2 (MRP2/ABCC2) and P-glycoprotein (P-gp/ABCB1) efflux transporters are governed by transporter-associated proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) formed by phosphatidylinositol 4-phosphate 5-kinase type 1 (PIP5K1) activates the linker function of radixin for efflux transporters. Radixin is involved in the plasma membrane localization of efflux transporters. We examined whether PIP5K1 could be a target for the modulation of transporter activities in hepatocytes and cancer cells. Methods: The effects of PIP5K1 depletion by siRNA in mouse primary hepatocytes, PANC1 human pancreatic carcinoma cells, and HepG2 human hepatocellular carcinoma cells on the intracellular accumulation of MRP2 and P-gp substrates were examined. Results: PIP5K1A depletion resulted in increased intracellular accumulation of carboxydichlorofluorescein, a MRP2 fluorescent substrate, in mouse primary hepatocytes, PANC1 cells, and HepG2 cells. In PANC1 and HepG2 cells, the transport activities of MRP2 were significantly decreased by PIP5K1C depletion. However, the transport activities of P-gp were unchanged by PIP5K1 depletion. PIP2 levels were unchanged between control and PIP5K1A- or PIP5K1C-depleted HepG2 cells. MRP2 mRNA levels showed few changes in HepG2 cells following PIP5K1A or PIP5K1C depletion. The expression of phosphorylated radixin was decreased by PIP5K1A and PIP5K1C depletion, although total radixin levels were unchanged. Conclusions: These data suggest that PIP5K1A and PIP5K1C could be target proteins for modulating MRP2 function, partly because of the resulting changes of the linker function of radixin.
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Papasotiriou, Ioannis, Panagiotis Apostolou, Dimitrios-Athanasios Ntanovasilis, Panagiotis Parsonidis, Daniar Osmonov, and Klaus-Peter Jünemann. "Study and detection of potential markers for predicting metastasis into lymph nodes in prostate cancer." Biomarkers in Medicine 14, no. 14 (October 2020): 1317–27. http://dx.doi.org/10.2217/bmm-2020-0372.
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Hormone-refractory prostate carcinoma has a different cell surface protein profile than hormone-sensitive prostate carcinoma, which provides migration ability and interactions with organs/tissues. Detection and association of these proteins with lymph node metastasis via lymphadenectomy might be beneficial for patients. Gene expression analysis in hormone-refractory and hormone-sensitive commercial cancer cell lines was performed and, after co-cultivation with osteoblasts or endothelial cells, knockdown experiments followed to validate potential biomarkers. “ Myeloid-associated differentiation markers, myosin 1b and phosphatidylinositol-4-phosphate-5-kinase type 1 alpha are implicated in metastasis”, their knockdown altered the expression of key regulators of endothelial-mesenchymal transition, invasion, motility and migration. In primary prostate tumors, these genes could be an indicator for future metastasis into lymph nodes.
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Jakobsen, Søren N., D. Grahame Hardie, Nick Morrice, and Hans E. Tornqvist. "5′-AMP-activated Protein Kinase Phosphorylates IRS-1 on Ser-789 in Mouse C2C12 Myotubes in Response to 5-Aminoimidazole-4-carboxamide Riboside." Journal of Biological Chemistry 276, no. 50 (October 11, 2001): 46912–16. http://dx.doi.org/10.1074/jbc.c100483200.
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Exercise is known to increase insulin sensitivity and is an effective form of treatment for the hyperglycemia observed in type 2 diabetes. Activation of 5′-AMP-activated protein kinase (AMPK) by 5-aminoimidazole-4-carboxamide riboside (AICAR), exercise, or electrically stimulated contraction leads to increased glucose transport in skeletal muscle. Here we report the first evidence of a direct interaction between AMPK and the most upstream component of the insulin-signaling cascade, insulin receptor substrate-1 (IRS-1). We find that AMPK rapidly phosphorylates IRS-1 on Ser-789 in cell-free assays as well as in mouse C2C12 myotubes incubated with AICAR. In the C2C12 myotubes activation of AMPK by AICAR matched the phosphorylation of IRS-1 on Ser-789. This phosphorylation correlates with a 65% increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity in C2C12 myotubes preincubated with AICAR. The binding of phosphatidylinositol 3-kinase to IRS-1 was not affected by AICAR. These results demonstrate the existence of an interaction between AMPK and early insulin signaling that could be of importance to our understanding of the potentiating effects of exercise on insulin signaling.
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Park, S., W. Lee, KH You, H. Kim, JM Suh, HK Chung, M. Shong, and OY Kwon. "Regulation of phosphatidylinositol-phosphate kinase IIgamma gene transcription by thyroid-stimulating hormone in thyroid cells." Journal of Molecular Endocrinology 26, no. 2 (April 1, 2001): 127–33. http://dx.doi.org/10.1677/jme.0.0260127.
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This study was performed to evaluate the effects of thyroid-stimulating hormone (TSH) on phosphatidylinositol-4-phosphate 5-kinase type IIgamma (PIPKIIgamma) gene expression in the thyrocytes of FRTL-5 cells. Although PIPKIIgamma mRNA was expressed constantly in the absence of added TSH, its expression increased remarkably in the presence of 10(-9) M TSH. This increase started within 6 h of the addition of TSH, and reached a maximum at 8 h. The mRNA expression properties of PIPKIIgamma in the cells were identified using inhibitors. Actinomycin D blocked PIPKIIgamma transcription strongly, while cycloheximide did not. In an experiment using 5,6-dichlo-1-beta-d -ribofuranosylbenzimidaxole, the half-life of PIPKIIgamma mRNA was approximately 6 h in the presence or absence of TSH, and it was not affected by the stability of the PIPKIIgamma mRNA. The effects of TSH on PIPKIIgamma gene expression were specific, and other growth factors examined (transferrin, insulin and hydrocortisone) did not alter its expression. It is possible that the mechanism of PIPKIIgamma gene expression is involved in the permissive effect of the TSH-cAMP cascade proper. Our results indicate, for the first time, that the expression of PIPKIIgamma is regulated transcriptionally by TSH in thyrocytes.
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Grey, Andrew, Qi Chen, Karen Callon, Xin Xu, Ian R. Reid, and Jill Cornish. "The Phospholipids Sphingosine-1-Phosphate and Lysophosphatidic Acid Prevent Apoptosis in Osteoblastic Cells via a Signaling Pathway Involving Gi Proteins and Phosphatidylinositol-3 Kinase." Endocrinology 143, no. 12 (December 1, 2002): 4755–63. http://dx.doi.org/10.1210/en.2002-220347.
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Abstract The naturally occurring phospholipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have recently emerged as bioactive compounds that exert mitogenic effects in many cell types, including osteoblasts. In the current study, we examined the ability of each of these compounds to influence osteoblast survival. Using terminal deoxynucleotidyl transferase-mediated deoxyuridine 5′-triphosphate nick-end labeling and DNA fragmentation assays, we found that both LPA and S1P dose-dependently inhibited (by at least 50% and 40%, respectively) the apoptosis induced by serum withdrawal in cultures of primary calvarial rat osteoblasts and SaOS-2 cells. The antiapoptotic effects were inhibited by pertussis toxin, wortmannin, and LY294002, implicating Gi proteins and phosphatidylinositol-3 kinase (PI-3 kinase) in the signaling pathway that mediates phospholipid-induced osteoblast survival. Specific inhibitors of p42/44 MAPK signaling did not block LPA- or S1P-induced osteoblast survival. LPA and S1P induced PI-3 kinase-dependent activation of p70 S6 kinase, but rapamycin, a specific inhibitor of p70 S6 kinase activation, did not prevent phospholipid-induced osteoblast survival. LPA and S1P also inhibited apoptosis in Swiss 3T3 fibroblastic cells in a Gi protein-dependent fashion. In fibroblastic cells, however, the antiapoptotic effects of S1P were sensitive to inhibition of both PI-3 kinase and p42/44 MAPK signaling, whereas those of LPA were partially abrogated by inhibitors of p42/44 MAPK signaling but not by PI-3 kinase inhibitors. These data demonstrate that LPA and S1P potently promote osteoblast survival in vitro, and that cell-type specificity exists in the antiapoptotic signaling pathways activated by phospholipids.
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Jones, Carol A., Suzanne E. Greer-Phillips, and Katherine A. Borkovich. "The Response Regulator RRG-1 Functions Upstream of a Mitogen-activated Protein Kinase Pathway Impacting Asexual Development, Female Fertility, Osmotic Stress, and Fungicide Resistance inNeurospora crassa." Molecular Biology of the Cell 18, no. 6 (June 2007): 2123–36. http://dx.doi.org/10.1091/mbc.e06-03-0226.
Full textAbstract:
Two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, Archaea, fungi, slime molds, and plants. Here, we characterize RRG-1, a response regulator protein from the filamentous fungus Neurospora crassa. The cell lysis phenotype of Δrrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, Δrrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, Δrrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in Δrrg-1 strains. OS-2-phosphate levels are also low in Δrrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1D921Nallele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway.
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Tan, Xiaojun, Narendra Thapa, Yihan Liao, Suyong Choi, and Richard A. Anderson. "PtdIns(4,5)P2 signaling regulates ATG14 and autophagy." Proceedings of the National Academy of Sciences 113, no. 39 (September 12, 2016): 10896–901. http://dx.doi.org/10.1073/pnas.1523145113.
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Autophagy is a regulated self-digestion pathway with fundamental roles in cell homeostasis and diseases. Autophagy is regulated by coordinated actions of a series of autophagy-related (ATG) proteins. The Barkor/ATG14(L)–VPS34 (a class III phosphatidylinositol 3-kinase) complex and its product phosphatidylinositol 3-phosphate [PtdIns(3)P] play key roles in autophagy initiation. ATG14 contains a C-terminal Barkor/ATG14(L) autophagosome-targeting sequence (BATS) domain that senses the curvature of PtdIns(3)P-containing membrane. The BATS domain also strongly binds PtdIns(4,5)P2, but the functional significance has been unclear. Here we show that ATG14 specifically interacts with type Iγ PIP kinase isoform 5 (PIPKIγi5), an enzyme that generates PtdIns(4,5)P2 in mammalian cells. Autophagosomes have associated PIPKIγi5 and PtdIns(4,5)P2 that are colocalized with late endosomes and the endoplasmic reticulum. PtdIns(4,5)P2 generation at these sites requires PIPKIγi5. Loss of PIPKIγi5 results in a loss of ATG14, UV irradiation resistance-associated gene, and Beclin 1 and a block of autophagy. PtdIns(4,5)P2 binding to the ATG14–BATS domain regulates ATG14 interaction with VPS34 and Beclin 1, and thus plays a key role in ATG14 complex assembly and autophagy initiation. This study identifies an unexpected role for PtdIns(4,5)P2 signaling in the regulation of ATG14 complex and autophagy.
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Jessen, Niels, Rasmus Pold, Esben S. Buhl, Lasse S. Jensen, Ole Schmitz, and Sten Lund. "Effects of AICAR and exercise on insulin-stimulated glucose uptake, signaling, and GLUT-4 content in rat muscles." Journal of Applied Physiology 94, no. 4 (April 1, 2003): 1373–79. http://dx.doi.org/10.1152/japplphysiol.00250.2002.
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Physical activity is known to increase insulin action in skeletal muscle, and data have indicated that 5′-AMP-activated protein kinase (AMPK) is involved in the molecular mechanisms behind this beneficial effect. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) can be used as a pharmacological tool to repetitively activate AMPK, and the objective of this study was to explore whether the increase in insulin-stimulated glucose uptake after either long-term exercise or chronic AICAR administration was followed by fiber-type-specific changes in insulin signaling and/or changes in GLUT-4 expression. Wistar rats were allocated into three groups: an exercise group trained on treadmill for 5 days, an AICAR group exposed to daily subcutaneous injections of AICAR, and a sedentary control group. AMPK activity, insulin-stimulated glucose transport, insulin signaling, and GLUT-4 expression were determined in muscles characterized by different fiber type compositions. Both exercised and AICAR-injected animals displayed a fiber-type-specific increase in glucose transport with the most marked increase in muscles with a high content of type IIb fibers. This increase was accompanied by a concomitant increase in GLUT-4 expression. Insulin signaling as assessed by phosphatidylinositol 3-kinase and PKB/Akt activity was enhanced only after AICAR administration and in a non-fiber-type-specific manner. In conclusion, chronic AICAR administration and long-term exercise both improve insulin-stimulated glucose transport in skeletal muscle in a fiber-type-specific way, and this is associated with an increase in GLUT-4 content.
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Heled, Yuval, Yair Shapiro, Yoav Shani, Dani S. Moran, Lea Langzam, Liora Braiman, Sanford R. Sampson, and Joseph Meyerovitch. "Physical exercise prevents the development of type 2 diabetes mellitus in Psammomys obesus." American Journal of Physiology-Endocrinology and Metabolism 282, no. 2 (February 1, 2002): E370—E375. http://dx.doi.org/10.1152/ajpendo.00296.2001.
Full textAbstract:
We hypothesized that exercise training might prevent diabetes mellitus in Psammomys obesus. Animals were assigned to three groups: high-energy diet (CH), high-energy diet and exercise (EH), and low-energy diet (CL). The EH group ran on a treadmill 5 days/wk, twice a day. After 4 wk, 93% of the CH group were diabetic compared with only 20% of the EH group. There was no difference in weight gain among the groups. Both EH and CH groups were hyperinsulinemic. Epididymal fat (% of body weight) was higher in the CH group than in either the EH and or the CL group. Protein kinase C (PKC)-δ activity and serine phosphorylation were higher in the EH group. No differences were found in tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase among the groups. We demonstrate for the first time that exercise training effectively prevents the progression of diabetes mellitus type 2 in Psammomys obesus. PKC-δ may be involved in the adaptive effects of exercise in skeletal muscles that lead to the prevention of type 2 diabetes mellitus.
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Ono, Hiraku, Hideki Katagiri, Makoto Funaki, Motonobu Anai, Kouichi Inukai, Yasushi Fukushima, Hideyuki Sakoda, et al. "Regulation of Phosphoinositide Metabolism, Akt Phosphorylation, and Glucose Transport by PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) in 3T3-L1 Adipocytes." Molecular Endocrinology 15, no. 8 (August 1, 2001): 1411–22. http://dx.doi.org/10.1210/mend.15.8.0684.
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Abstract To investigate the roles of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the regulation of 3-position phosphorylated phosphoinositide metabolism as well as insulin-induced Akt phosphorylation and glucose metabolism, wild-type PTEN and its phosphatase-dead mutant (C124S) with or without an N-terminal myristoylation tag were overexpressed in Sf-9 cells and 3T3-L1 adipocytes using baculovirus and adenovirus systems, respectively. When expressed in Sf-9 cells together with the p110α catalytic subunit of phosphoinositide 3-kinase, myristoylated PTEN markedly reduced the accumulations of both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate induced by p110α. In contrast, overexpression of the C124S mutants apparently increased these accumulations. In 3T3-L1 adipocytes, insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate were markedly suppressed by overexpression of wild-type PTEN with the N-terminal myristoylation tag, but not by that without the tag. On the contrary, the C124S mutants of PTEN enhanced insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Interestingly, the phosphorylation level of Akt at Thr308 (Akt2 at Thr309), but not at Ser473 (Akt2 at Ser474), was revealed to correlate well with the accumulation of phosphatidylinositol 3,4,5-trisphosphate modified by overexpression of these PTEN proteins. Finally, insulin-induced increases in glucose transport activity were significantly inhibited by the overexpression of myristoylated wild-type PTEN, but were not enhanced by expression of the C124S mutant of PTEN. Therefore, in conclusion, 1) PTEN dephosphorylates both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in vivo, and the C124S mutants interrupt endogenous PTEN activity in a dominant-negative manner. 2) The membrane targeting process of PTEN may be important for exerting its function. 3) Phosphorylations of Thr309 and Ser474 of Akt2 are regulated differently, and the former is regulated very sensitively by the function of PTEN. 4) The phosphorylation level of Ser474, but not that of Thr309, in Akt2 correlates well with insulin-stimulated glucose transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous PTEN may not play a major role in the regulation of glucose transport activity in 3T3-L1 adipocytes.
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Dantas, Ana Paula V., Junsuke Igarashi, and Thomas Michel. "Sphingosine 1-phosphate and control of vascular tone." American Journal of Physiology-Heart and Circulatory Physiology 284, no. 6 (June 1, 2003): H2045—H2052. http://dx.doi.org/10.1152/ajpheart.01089.2002.
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Sphingosine-1-phosphate (S1P) is a platelet-derived lipid mediator that activates the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells. However, the role of S1P in endothelium-dependent vasodilation and the signaling pathways elicited by S1P in intact vessels are largely unknown. We found that S1P induces dose-dependent transient relaxation of isolated pressurized mesenteric arterioles (EC5010 ± 3 nM); maximal vasodilation (55 ± 8%) is seen ∼2 min after S1P addition and returns to baseline by 5 min. S1P promotes comparable responses in arterioles from wild-type but not eNOSnullmice. S1P-induced vasodilation is abrogated by removal of endothelium or by the addition of the NOS inhibitor Nω-monomethyl-l-arginine but is not affected by the cyclooxygenase inhibitor indomethacin, nor by the blockade of K+channels by using 4-aminopyridine. S1P-induced vasodilation is attenuated by pertussis toxin, by the phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin, and by the calcium chelator BAPTA. With the use of high-sensitivity protein immunoblots in extracts from single pressurized vessels, we found that S1P, but not bradykinin, promotes the phosphorylation of eNOS at Ser1179. Maximum S1P-induced eNOS Ser1179phosphorylation was reached at the time of maximum vasorelaxation, but enzyme phosphorylation persisted for several minutes after vasodilation had resolved. Thus regulatory pathways distinct from eNOS Ser1179dephosphorylation serve to terminate agonist-promoted vasorelaxation. Taken together, our findings demonstrate that S1P, an important intercellular mediator of platelet-vessel wall interactions, is a effective arteriolar vasodilator that acts via G protein-dependent, calcium-sensitive, and PI3-kinase-modulated signaling pathways.
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Shi, Min, Michael L. Mathai, Guoqin Xu, Xiao Q. Su, and Andrew J. McAinch. "The effect of dietary supplementation with blueberry, cyanidin-3-O-β-glucoside, yoghurt and its peptides on gene expression associated with glucose metabolism in skeletal muscle obtained from a high-fat-high-carbohydrate diet induced obesity model." PLOS ONE 17, no. 9 (September 16, 2022): e0270306. http://dx.doi.org/10.1371/journal.pone.0270306.
Full textAbstract:
Obesity is a leading global health problem contributing to various chronic diseases, including type II diabetes mellitus (T2DM). The aim of this study was to investigate whether blueberries, yoghurt, and their respective bioactive components, Cyanidin-3-O-β-glucoside (C3G) and peptides alone or in combinations, alter the expression of genes related to glucose metabolism in skeletal muscles from diet-induced obese mice. In extensor digitorum longus (EDL), yoghurt up-regulated the expression of activation of 5’adenosine monophosphate-activated protein kinase (AMPK), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3 kinase (PI3K) and glucose transporter 4 (GLUT4), and down-regulated the expression of angiotensin II receptor type 1 (AGTR-1). The combination of blueberries and yoghurt down-regulated the mRNA expression of AGTR-1 and Forkhead box protein O1 (FoxO1) in the EDL. Whereas the combination of C3G and peptides down-regulated AGTR-1 and up-regulated GLUT4 mRNA expression in the EDL. In the soleus, blueberries and yoghurt alone, and their combination down-regulated AGTR-1 and up-regulated GLUT4 mRNA expression. In summary blueberries and yoghurt, regulated multiple genes associated with glucose metabolism in skeletal muscles, and therefore may play a role in the management and prevention of T2DM.
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Farese, Robert V., Mini P. Sajan, and Mary L. Standaert. "Insulin-Sensitive Protein Kinases (Atypical Protein Kinase C and Protein Kinase B/Akt): Actions and Defects in Obesity and Type II Diabetes." Experimental Biology and Medicine 230, no. 9 (October 2005): 593–605. http://dx.doi.org/10.1177/153537020523000901.
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Glucose transport into muscle is the initial process in glucose clearance and is uniformly defective in insulin-resistant conditions of obesity, metabolic syndrome, and Type II diabetes mellitus. Insulin regulates glucose transport by activating insulin receptor substrate-1 (IRS-1)-dependent phosphatidylinositol 3-kinase (PI3K) which, via increases in PI-3, 4, 5-triphosphate (PIP3), activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). Here, we review (i) the evidence that both aPKC and PKB are required for insulin-stimulated glucose transport, (ii) abnormalities in muscle aPKC/PKB activation seen in obesity and diabetes, and (iii) mechanisms for impaired aPKC activation in insulin-resistant conditions. In most cases, defective muscle aPKC/PKB activation reflects both impaired activation of IRS-1/PI3K, the upstream activator of aPKC and PKB in muscle and, in the case of aPKC, poor responsiveness to PIP3, the lipid product of PI3K. Interestingly, insulin-sensitizing agents (e.g., thiazolidinediones, metformin) improve aPKC activation by insulin in vivo and PIP3 in vitro, most likely by activating 5′-adenosine monophosphate-activated protein kinase, which favorably alters intracellular lipid metabolism. Differently from muscle, aPKC activation in the liver is dependent on IRS-2/PI3K rather than IRS-1/PI3K and, surprisingly, the activation of IRS-2/PI3K and aPKC is conserved in high-fat feeding, obesity, and diabetes. This conservation has important implications, as continued activation of hepatic aPKC in hyperinsulinemic states may increase the expression of sterol regulatory element binding protein-1c, which controls genes that increase hepatic lipid synthesis. On the other hand, the defective activation of IRS-1/PI3K and PKB, as seen in diabetic liver, undoubtedly and importantly contributes to increases in hepatic glucose output. Thus, the divergent activation of aPKC and PKB in the liver may explain why some hepatic actions of insulin (e.g., aPKC-dependent lipid synthesis) are increased while other actions (e.g., PKB-dependent glucose metabolism) are diminished. This may explain the paradox that the liver secretes excessive amounts of both very low density lipoprotein triglycerides and glucose in Type II diabetes. Previous reviews from our laboratory that have appeared in the Proceedings have provided essentials on phospholipid-signaling mechanisms used by insulin to activate several protein kinases that seem to be important in mediating the metabolic effects of insulin. During recent years, there have been many new advances in our understanding of how these lipid-dependent protein kinases function during insulin action and why they fail to function in states of insulin resistance. The present review will attempt to summarize what we believe are some of the more important advances.
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Javaux, F., M. F. Vincent, D. R. Wagner, and G. van den Berghe. "Cell-type specificity of inhibition of glycolysis by 5-amino-4-imidazolecarboxamide riboside. Lack of effect in rabbit cardiomyocytes and human erythrocytes, and inhibition in FTO-2B rat hepatoma cells." Biochemical Journal 305, no. 3 (February 1, 1995): 913–19. http://dx.doi.org/10.1042/bj3050913.
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The nucleoside AICAriboside (5-amino-4-imidazolecarboxamide riboside) has been shown to inhibit glycolysis in isolated rat hepatocytes [Vincent, Bontemps and Van den Berghe (1992) Biochem. J. 281, 267-272]. The effect is mediated by AICA-ribotide (ZMP), the product of the phosphorylation of AICA-riboside by adenosine kinase. To assess the cell-type specificity of the effect, studies were conducted in rabbit cardiomyocytes, human erythrocytes and rat hepatoma FTO-2B cells. AICA-riboside had no effect on glycolysis in cardiomyocytes, and a slight stimulatory effect in erythrocytes, but inhibited glycolysis by 65% at 250 microM concentration in FTO-2B cells, although only when tissue-culture medium was replaced by Krebs-Ringer bicarbonate buffer. At 500 microM AICAriboside, ZMP remained undetectable in cardiomyocytes, but reached 0.65 mM in erythrocytes and 5 mM in FTO-2B cells. In the latter, AICAriboside provoked up to 2-fold elevations of glucose 6-phosphate and fructose 6-phosphate, accompanied by a decrease in fructose 1,6-bisphosphate. This indicated inhibition of 6-phosphofructo-1-kinase (PFK-1). Accordingly, in FTO-2B cell-free extracts, the activity of PFK-1, measured under physiological conditions, was inhibited by approx. 70% by 5 mM ZMP. ZMP had a less pronounced effect on the activity of PFK-1 in normal rat liver; it did not influence the activity of PFK-1 in rat muscle, rabbit heart and human erythrocytes. It is concluded that the inhibitory effect of AICAriboside on glycolysis is dependent on both (1) the capacity of the cells to accumulate ZMP and (2) the presence of target enzymes which are sensitive to ZMP.
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Bonangelino, C. J., N. L. Catlett, and L. S. Weisman. "Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology." Molecular and Cellular Biology 17, no. 12 (December 1997): 6847–58. http://dx.doi.org/10.1128/mcb.17.12.6847.
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During cell division, the vacuole of Saccharomyces cerevisiae partitions between mother and daughter cells. A portion of the parental vacuole membrane moves into the bud, and ultimately membrane scission divides the vacuole into two separate structures. Here we characterize two yeast mutations causing defects in vacuole membrane scission, vac7-1 and vac14-1. A third mutant, afab1-2 strain, isolated in a nonrelated screen (A. Yamamoto et al., Mol. Biol. Cell 6:525-539, 1995) shares the vacuolar phenotypes of the vac7-1 and vac14-1 strains. Unlike the wild type, mutant vacuoles are not multilobed structures; in many cases, a single vacuole spans both the mother and bud, with a distinct gap in the mother-bud neck. Thus, even where the membranes are closely opposed, vacuole fission is arrested. Simply enlarging the vacuole does not produce this mutant phenotype. An additional common phenotype of these mutants is a defect in vacuole acidification; however, vacuole scission in most other vacuole acidification mutants is normal. An alteration in vacuole membrane lipids could account for both the vacuole membrane scission and acidification defects. Because a directed screen has not identified additional class III complementation groups, it is likely that all three genes are involved in a similar process. Interestingly, FAB1, was previously shown to encode a putative phosphatidylinositol-4-phosphate 5-kinase. Moreover, overexpression of FAB1 suppresses the vac14-1 mutation, which suggests that VAC14 and FAB1 act at a common step. VAC7 encodes a novel 128-kDa protein that is localized at the vacuole membrane. This location of Vac7p is consistent with its involvement in vacuole morphology and inheritance.
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Lee, Myung-Ja, Denis Feliers, Meenalakshmi M. Mariappan, Kavithalakshmi Sataranatarajan, Lenin Mahimainathan, Nicolas Musi, Marc Foretz, et al. "A role for AMP-activated protein kinase in diabetes-induced renal hypertrophy." American Journal of Physiology-Renal Physiology 292, no. 2 (February 2007): F617—F627. http://dx.doi.org/10.1152/ajprenal.00278.2006.
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We tested the hypothesis that AMP-activated protein kinase (AMPK), an energy sensor, regulates diabetes-induced renal hypertrophy. In kidney glomerular epithelial cells, high glucose (30 mM), but not equimolar mannitol, stimulated de novo protein synthesis and induced hypertrophy in association with increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2, regulatory events in mRNA translation. These high-glucose-induced changes in protein synthesis were phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR) dependent and transforming growth factor-β independent. High glucose reduced AMPK α-subunit theronine (Thr) 172 phosphorylation, which required Akt activation. Changes in AMP and ATP content could not fully account for high-glucose-induced reductions in AMPK phosphorylation. Metformin and 5-aminoimidazole-4-carboxamide-1β-riboside (AICAR) increased AMPK phosphorylation, inhibited high-glucose stimulation of protein synthesis, and prevented high-glucose-induced changes in phosphorylation of 4E binding protein 1 and eukaryotic elongation factor 2. Expression of kinase-inactive AMPK further increased high-glucose-induced protein synthesis. Renal hypertrophy in rats with Type 1 diabetes was associated with reduction in AMPK phosphorylation and increased mTOR activity. In diabetic rats, metformin and AICAR increased renal AMPK phosphorylation, reversed mTOR activation, and inhibited renal hypertrophy, without affecting hyperglycemia. AMPK is a newly identified regulator of renal hypertrophy in diabetes.
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Egom, Emmanuel Eroume A., Tamer M. A. Mohamed, Mamas A. Mamas, Ying Shi, Wei Liu, Debora Chirico, Sally E. Stringer, et al. "Activation of Pak1/Akt/eNOS signaling following sphingosine-1-phosphate release as part of a mechanism protecting cardiomyocytes against ischemic cell injury." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 4 (October 2011): H1487—H1495. http://dx.doi.org/10.1152/ajpheart.01003.2010.
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We investigated whether plasma long-chain sphingoid base (LCSB) concentrations are altered by transient cardiac ischemia during percutaneous coronary intervention (PCI) in humans and examined the signaling through the sphingosine-1-phosphate (S1P) cascade as a mechanism underlying the S1P cardioprotective effect in cardiac myocytes. Venous samples were collected from either the coronary sinus ( n = 7) or femoral vein ( n = 24) of 31 patients at 1 and 5 min and 12 h, following induction of transient myocardial ischemia during elective PCI. Coronary sinus levels of LCSB were increased by 1,072% at 1 min and 941% at 5 min ( n = 7), while peripheral blood levels of LCSB were increased by 579% at 1 min, 617% at 5 min, and 436% at 12 h ( n = 24). In cultured cardiac myocytes, S1P, sphingosine (SPH), and FTY720, a sphingolipid drug candidate, showed protective effects against CoCl induced hypoxia/ischemic cell injury by reducing lactate dehydrogenase activity. Twenty-five nanomolars of FTY720 significantly increased phospho-Pak1 and phospho-Akt levels by 56 and 65.6% in cells treated with this drug for 15 min. Further experiments demonstrated that FTY720 triggered nitric oxide release from cardiac myocytes is through pertussis toxin-sensitive phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase signaling. In ex vivo hearts, ischemic preconditioning was cardioprotective in wild-type control mice (Pak1f/f), but this protection appeared to be ineffective in cardiomyocyte-specific Pak1 knockout (Pak1cko) hearts. The present study provides the first direct evidence of the behavior of plasma sphingolipids following transient cardiac ischemia with dramatic and early increases in LCSB in humans. We also demonstrated that S1P, SPH, and FTY720 have protective effects against hypoxic/ischemic cell injury, likely a Pak1/Akt1 signaling cascade and nitric oxide release. Further study on a mouse model of cardiac specific deletion of Pak1 demonstrates a crucial role of Pak1 in cardiac protection against ischemia/reperfusion injury.
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Canabal, Debra D., Joseph G. Potian, Ricardo G. Duran, Joseph J. McArdle, and Vanessa H. Routh. "Hyperglycemia impairs glucose and insulin regulation of nitric oxide production in glucose-inhibited neurons in the ventromedial hypothalamus." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 293, no. 2 (August 2007): R592—R600. http://dx.doi.org/10.1152/ajpregu.00207.2007.
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Physiological changes in extracellular glucose, insulin, and leptin regulate glucose-excited (GE) and glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH). Nitric oxide (NO) signaling, which is involved in the regulation of food intake and insulin signaling, is altered in obesity and diabetes. We previously showed that glucose and leptin inhibit NO production via the AMP-activated protein kinase (AMPK) pathway, while insulin stimulates NO production via the phosphatidylinositol-3-OH kinase (PI3K) pathway in VMH GI neurons. Hyperglycemia-induced inhibition of AMPK reduces PI3K signaling by activating the mammalian target of rapamycin (mTOR). We hypothesize that hyperglycemia impairs glucose and insulin-regulated NO production in VMH GI neurons. This hypothesis was tested in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced type 1 diabetic rats using NO- and membrane potential-sensitive dyes. Neither decreased extracellular glucose from 2.5 to 0.5 mM, nor 5 nM insulin increased NO production in VMH neurons in either experimental condition. Glucose- and insulin-regulated NO production was restored in the presence of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside or the mTOR inhibitor rapamycin. Finally, decreased glucose and insulin did not alter membrane potential in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced rats. These data suggest that hyperglycemia impairs glucose and insulin regulation of NO production through AMPK inhibition. Furthermore, glucose and insulin signaling pathways interact via the mTOR pathway.
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McEwen, Scott T., Sarah F. Balus, Matthew J. Durand, and Julian H. Lombard. "Angiotensin II maintains cerebral vascular relaxation via EGF receptor transactivation and ERK1/2." American Journal of Physiology-Heart and Circulatory Physiology 297, no. 4 (October 2009): H1296—H1303. http://dx.doi.org/10.1152/ajpheart.01325.2008.
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This study identified, on the integrative level, two components of the ANG II signaling pathway that lay downstream from the ANG II type 1 (AT1) receptor and are critically involved in maintaining vascular relaxation in cerebral resistance arteries. In these experiments, the relaxation of isolated middle cerebral arteries (MCA) in response to ACh (10−9-10−5 M), iloprost (10−16-10−11 g/ml), and reduced PO2 was lost and the ratio of phospho-ERK/ERK1/2 was significantly reduced in aortas of male Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) diet to suppress plasma ANG II levels. In salt-fed rats, relaxation of MCA in response to these vasodilator stimuli was restored by chronic (3 days) intravenous infusion of either ANG II (5 ng·kg−1·min−1) or epidermal growth factor (EGF; 2 μg/h). The protective effect of ANG II infusion to restore vascular relaxation was eliminated by coinfusion of either the EGF receptor kinase inhibitor AG-1478 (20 μg/h), the ERK1/2 inhibitor PD-98059 (10 μg/h), or the protein synthesis inhibitor cycloheximide (5 μg/h). In rats fed a low-salt (0.4% NaCl) diet, MCA relaxation in response to ACh, reduced PO2, and iloprost was eliminated by intravenous infusion of AG-1478, PD-98059, or cycloheximide. In ANG II-infused rats fed HS diet, and in rats fed LS diet, vasodilator responses to reduced PO2 and iloprost were unaffected by the p38 MAP kinase inhibitor SB-203580 and the phosphatidylinositol 3-kinase inhibitor wortmannin. These findings indicate that maintenance of normal vascular relaxation mechanisms by ANG II in rat MCA requires activation of the EGF receptor kinase and ERK1/2.
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Kushnir, O. Yu, and I. M. Yaremii. "AGE-RELATED CHANGES OF GLYCOLYTIC ACTIVITY AND ANTIOXIDANT CAPACITY IN THE BLOOD OF ALLOXAN DIABETIC RATS." Актуальні проблеми сучасної медицини: Вісник Української медичної стоматологічної академії 20, no. 3 (November 12, 2020): 169–73. http://dx.doi.org/10.31718/2077-1096.20.3.169.
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The increasing incidence of type 1 diabetes coupled with advances in treatment of type 1 diabetes has resulted in an unprecedented number of older adults living with and controllable type 1 diabetes. The objective of this experimental study was to assess the impact of aging on the level of basal glycaemia and activities of glucose-6-phosphate dehydrogenase [EC1.1.1.49], pyruvate kinase [EC 2.7.1.40] and glutathione reductase [EC1.6.4.2] in erythrocytes of alloxan-diabetic rats. Methods: We used 100 male Wistar rats, divided into two age groups: I group included- 2-month (adult) animals, and II group was made up of 4-month (old) animals. Diabetes was modelled by injecting the rats with 5% solution of alloxan monohydrate intraperitoneally in a dose of 170 mg/kg. Blood was taken from the tail vein to evaluate the basal glycaemia on 5-th and 47-th day after the alloxan injection. Rats were sacrificed on the 47-th day of the experiment in accordance with the regulations on ethical treatment of vertebrates. The assessment of the activity of the enzymes was carried out by standard methods. Statistical analysis was performed by using Statistica 10 StatSoft Inc. Results. The level of basal glycaemia on the fifth day of the experiment in the animals of both groups went up on average by 115% from baseline values. We founded that on 47-th day this index was higher in group of old rats by 20% than in adult rats. Pyruvate kinase activity in erythrocytes of adult and old animals with diabetes decreased by 35% and 50% respectively compared with the control. Glucose-6-phosphate dehydrogenase activity in erythrocytes of adult and old animals with diabetes decreased by 27% and 45% respectively compared with the control on 47-th day. The changes may be considered as the result of age-related disorders of glucose metabolism due to disturbances in free radical mechanisms. Glutathione reductase activity in erythrocytes of adult and old animals with diabetes decreased by 29% and 35% respectively compared with the control on 47-th day. Conclusion. We have determined when getting aged, the alloxan-diabetic rats demonstrate changes in the sensitivity of pyruvate kinase, glucose-6-phosphate dehydrogenase and glutathione reductase activities in erythrocytes resulted from the effect of diabetes mellitus factors (hyperglycaemia). We can suggest that glycaemic control is key purpose for older patients with type 1 diabetes in order to prevent of complication, which can be aggravated with age.
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Munnik, T., R. F. Irvine, and A. Musgrave. "Rapid turnover of phosphatidylinositol 3-phosphate in the green alga Chlamydomonas eugametos: signs of a phosphatidylinositide 3-kinase signalling pathway in lower plants?" Biochemical Journal 298, no. 2 (March 1, 1994): 269–73. http://dx.doi.org/10.1042/bj2980269.
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When Chlamydomonas eugametos gametes were incubated in carrier-free [32P]P1, the label was rapidly incorporated into PtdInsP and PtdInsP2 and, after reaching a maximum within minutes, was chased out by recirculating unlabelled P1 in the cell. This pulse-chase labelling pattern reflects their rapid turnover. In contrast, 32P incorporation into the structural lipids was slow and continued for hours. Of the radioactivity in the PtdInsP spot, 15% was in PtdIns3P and the rest in PtdIns4P, and of that in the PtdInsP2 spot, 1% was in PtdIns(3,4)P2 and the rest in PtdIns(4,5)P2, confirming the findings by Irvine, Letcher, Stephens and Musgrave [(1992) Biochem. J. 281, 269-266]. When cells were labelled with carrier-free [32P]P1, both PtdInsP isomers incorporated label in a pulse-chase-type pattern, demonstrating for the first time in a plant or animal system that D-3 poly-phosphoinositides turn over rapidly in non-stimulated cells, with kinetics similar to those shown by the D-4 isomers. In animal systems such lipids are already established as signalling molecules, and the data suggest that a similar role must be sought for them in lower plants such as Chlamydomonas.
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Chavakis, Triantafyllos, Antje Willuweit, Florea Lupu, Klaus Preissner, and Sandip Kanse. "Release of Soluble Urokinase Receptor from Vascular Cells*." Thrombosis and Haemostasis 86, no. 08 (2001): 686–93. http://dx.doi.org/10.1055/s-0037-1616105.
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SummaryUrokinase-type plasminogen activator (uPA) and its cell surface-receptor (uPAR) regulate cellular functions linked to adhesion and migration and contribute to pericellular proteolysis in tissue remodelling processes. Soluble uPAR (suPAR) is present in the circulation, peritoneal and ascitic fluid and in the cystic fluid from ovarian cancer. We have investigated the origin and the vascular distribution of the soluble receptor, which accounts for 10-20% of the total receptor in vascular endothelial and smooth muscle cells. Phase separation analysis of the cell conditioned media with Triton X-114 indicated that suPAR associates with the aqueous phase, indicative of the absence of the glycolipid anchor. There was a polarized release of suPAR from cultured endothelial cells towards the basolateral direction, whereas the membrane-bound receptor was found preferentially on the apical surface. Both, uPAR and suPAR became upregulated 2-4 fold after activation of protein kinase C with phorbol ester, which required de-novo protein biosynthesis. Interleukin-1β (IL-1β), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor increased suPAR release from endothelial cells, whereas platelet derived growth factor-BB, bFGF or IL-1β stimulated suPAR release from vascular smooth muscle cells. Immune electron microscopy indicated that in atherosclerotic vessels (s)uPAR was observed on cell membranes as well as in the extracellular matrix. These findings indicate that (s)uPAR from vascular cells is upregulated by proangiogenic as well as proatherogenic growth factors and cytokines, is preferentially released towards the basolateral side of endothelial cells and accumulates in the vessel wall.*Part of this work was supported by grants (Pr 327/1-4) from the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the Novartis-Foundation (Nürnberg, Germany). This work is part of the MD/PhD-thesis of T.C. at the Institute for Biochemistry, Department of Medicine, Justus-Liebig-Universität, Giessen, Germany. Abbreviations: bFGF: basic fibroblast growth factor, GPI: glycosyl-phosphatidylinositol, FCS: fetal calf serum, HUVEC: human umbilical vein endothelial cells, HVSMC: human vascular smooth muscle cells, IL-1β: interleukin-1β, mAb: monoclonal antibody, PBS: phosphate-buffered saline, PDGF-BB: platelet derived growth factor-BB, piPLC: phosphatidylinositol-specific phospholipase C, piPLD: phosphatidylinositol-specific phospholipase D, PMA: phorbol myristate acetate, scuPA: single chain uPA, suPAR: soluble urokinase receptor, uPA: urokinase- type plasminogen activator, uPAR: urokinase receptor, VN: vitronectin
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Friedlaender, M. M., D. Jain, Z. Ahmed, D. Hart, R. L. Barnett, and E. P. Nord. "Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+." American Journal of Physiology-Renal Physiology 264, no. 5 (May 1, 1993): F845—F853. http://dx.doi.org/10.1152/ajprenal.1993.264.5.f845.
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Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.
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Ford, Christopher P., Kenneth V. Wong, Van B. Lu, Elena Posse de Chaves, and Peter A. Smith. "Differential Neurotrophic Regulation of Sodium and Calcium Channels in an Adult Sympathetic Neuron." Journal of Neurophysiology 99, no. 3 (March 2008): 1319–32. http://dx.doi.org/10.1152/jn.00966.2007.
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Adult neuronal phenotype is maintained, at least in part, by the sensitivity of individual neurons to a specific selection of neurotrophic factors and the availability of such factors in the neurons' environment. Nerve growth factor (NGF) increases the functional expression of Na+ channel currents ( INa) and both N- and L-type Ca2+ currents ( ICa,N and ICa,L) in adult bullfrog sympathetic ganglion (BFSG) B-neurons. The effects of NGF on ICa involve the mitogen-activated protein kinase (MAPK) pathway. Prolonged exposure to the ganglionic neurotransmitter luteinizing hormone releasing hormone (LHRH) also increases ICa,N but the transduction mechanism remains to be elucidated as does the transduction mechanism for NGF regulation of Na+ channels. We therefore exposed cultured BFSG B-neurons to chicken II LHRH (0.45 μM; 6–9 days) or to NGF (200 ng/ml; 9–10 days) and used whole cell recording, immunoblot analysis, and ras or rap-1 pulldown assays to study effects of various inhibitors and activators of transduction pathways. We found that 1) LHRH signals via ras-MAPK to increase ICa,N, 2) this effect is mediated via protein kinase C-β (PKC-β-ΙΙ), 3) protein kinase A (PKA) is necessary but not sufficient to effect transduction, 4) NGF signals via phosphatidylinositol 3-kinase (PI3K) to increase INa, and 5) long-term exposure to LHRH fails to affect INa. Thus downstream signaling from LHRH has access to the ras-MAPK pathway but not to the PI3K pathway. This allows for differential retrograde and anterograde neurotrophic regulation of sodium and calcium channels in an adult sympathetic neuron.
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Bulhak, Aliaksandr A., Christian Jung, Claes-Göran Östenson, Jon O. Lundberg, Per-Ove Sjöquist, and John Pernow. "PPAR-α activation protects the type 2 diabetic myocardium against ischemia-reperfusion injury: involvement of the PI3-Kinase/Akt and NO pathway." American Journal of Physiology-Heart and Circulatory Physiology 296, no. 3 (March 2009): H719—H727. http://dx.doi.org/10.1152/ajpheart.00394.2008.
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Several clinical studies have shown the beneficial cardiovascular effects of fibrates in patients with diabetes and insulin resistance. The ligands of peroxisome proliferator-activated receptor-α (PPAR-α) reduce ischemia-reperfusion injury in nondiabetic animals. We hypothesized that the activation of PPAR-α would exert cardioprotection in type 2 diabetic Goto-Kakizaki (GK) rats, involving mechanisms related to nitric oxide (NO) production via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. GK rats and age-matched Wistar rats (n ≥ 7) were given either 1) the PPAR-α agonist WY-14643 (WY), 2) dimethyl sulfoxide (DMSO), 3) WY and the NO synthase inhibitor NG-nitro-l-arginine (l-NNA), 4) l-NNA, 5) WY and the PI3K inhibitor wortmannin, or 6) wortmannin alone intravenously before a 35-min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), expression of endothelial NO synthase (eNOS), inducible NO synthase, and Akt as well as nitrite/nitrate were determined. The IS was 75 ± 3% and 72 ± 4% of the area at risk in the Wistar and GK DMSO groups, respectively. WY reduced IS to 56 ± 3% in Wistar ( P < 0.05) and to 46 ± 5% in GK rats ( P < 0.001). The addition of either l-NNA or wortmannin reversed the cardioprotective effect of WY in both Wistar (IS, 70 ± 5% and 65 ± 5%, respectively) and GK (IS, 66 ± 4% and 64 ± 4%, P < 0.05, respectively) rats. The expression of eNOS and eNOS Ser1177 in the ischemic myocardium from both strains was increased after WY. The expression of Akt, Akt Ser473, and Akt Thr308 was also increased in the ischemic myocardium from GK rats following WY. Myocardial nitrite/nitrate levels were reduced in GK rats ( P < 0.05). The results suggest that PPAR-α activation protects the type 2 diabetic rat myocardium against ischemia-reperfusion injury via the activation of the PI3K/Akt and NO pathway.
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YAMADA, Kazuya, and Tamio NOGUCHI. "Nutrient and hormonal regulation of pyruvate kinase gene expression." Biochemical Journal 337, no. 1 (December 17, 1998): 1–11. http://dx.doi.org/10.1042/bj3370001.
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Mammalian pyruvate kinase (PK), a key glycolytic enzyme, has two genes named PKL and PKM, which produce the L- and R-type isoenzymes by means of alternative promoters, and the M1-and M2-types by mutually exclusive alternative splicing respectively. The expression of these genes is tissue-specific and under developmental, dietary and hormonal control. The L-type isoenzyme (L-PK) gene contains multiple regulatory elements necessary for regulation in the 5´ flanking region, up to position -170. Both L-II and L-III elements are required for stimulation of L-PK gene transcription by carbohydrates such as glucose and fructose, although the L-III element is itself responsive to carbohydrates. The L-II element is also responsible for the gene regulation by polyunsaturated fatty acids. Nuclear factor-1 proteins and hepatocyte nuclear factor 4, which bind to the L-II element, may also be involved in carbohydrate and polyunsaturated fatty acid regulation of the L-PK gene respectively. However, the L-III-element-binding protein that is involved in carbohydrate regulation remains to be clarified, although involvement by an upstream stimulating factor has been proposed. Available evidence suggests that the carbohydrate signalling pathway to the L-PK gene includes a glucose metabolite, possibly glucose 6-phosphate or xylulose 5-phosphate, as well as phosphorylation and dephosphorylation mechanisms. In addition, at least five regulatory elements have been identified in the 5´ flanking region of the PKM gene up to position -279. Sp1-family proteins bind to two proximal elements, but the binding of proteins to other elements have not yet been clarified. Glucose may stimulate the transcription of the PKM gene via hexosamine derivatives. Sp1 may be involved in this regulation via its dephosphorylation, although the carbohydrate response element has not been determined precisely in the PKM gene. Thus glucose stimulates transcription of the PKM gene by the mechanism which is probably different from the L-PK gene.
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GHELLI, Anna, Anna M. PORCELLI, Annalisa FACCHINI, Silvana HRELIA, Flavio FLAMIGNI, and Michela RUGOLO. "Phospholipase D1 is threonine-phosphorylated in human-airway epithelial cells stimulated by sphingosine-1-phosphate by a mechanism involving Src tyrosine kinase and protein kinase Cδ." Biochemical Journal 366, no. 1 (August 15, 2002): 187–93. http://dx.doi.org/10.1042/bj20020264.
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The regulatory role of protein kinase C (PKC) δ isoform in the stimulation of phospholipase D (PLD) by sphingosine-1-phosphate (SPP) in a human-airway epithelial cell line (CFNPE9o−) was revealed by using antisense oligodeoxynucleotide to PKCδ, in combination with the specific inhibitor rottlerin. Cell treatment with antisense oligodeoxynucleotide, but not with sense oligodeoxynucleotide, completely eliminated PKCδ expression and resulted in the strong inhibition of SPP-stimulated phosphatidic acid formation. Indeed, among the PKCα, β, δ, ∊ and ζ isoforms expressed in these cells, only PKCδ was activated on cell stimulation with SPP, as indicated by translocation into the membrane fraction. Furthermore, pertussis toxin and genistein eliminated both PKCδ translocation and PLD activation. In particular, a significant reduction in phosphatidylbutanol formation by SPP was observed in the presence of 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP1), an inhibitor of Src tyrosine kinase. Furthermore, the activity of Src kinase was slightly increased by SPP and inhibited by PP1. However, the level of PKCδ tyrosine phosphorylation was not increased in SPP-stimulated cells, suggesting that Src did not directly phosphorylate PKCδ. Finally, the level of serine phosphorylation of PLD1 and PLD2 isoforms was not changed, whereas the PLD1 isoform alone was threonine-phosphorylated in SPP-treated cells. PLD1 threonine phosphorylation was strongly inhibited by rottlerin, by anti-PKCδ oligodeoxynucleotide and by PP1. In conclusion, in CFNPE9o− cells, SPP interacts with a membrane receptor linked to a Gi type of G-protein, leading to activation of PLD, probably the PLD1 isoform, by a signalling pathway involving Src and PKCδ.
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Lamia, Katja A., Odile D. Peroni, Young-Bum Kim, Lucia E. Rameh, Barbara B. Kahn, and Lewis C. Cantley. "Increased Insulin Sensitivity and Reduced Adiposity in Phosphatidylinositol 5-Phosphate 4-Kinase β−/− Mice." Molecular and Cellular Biology 24, no. 11 (June 1, 2004): 5080–87. http://dx.doi.org/10.1128/mcb.24.11.5080-5087.2004.
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ABSTRACT Phosphorylated derivatives of the lipid phosphatidylinositol are known to play critical roles in insulin response. Phosphatidylinositol 5-phosphate 4-kinases convert phosphatidylinositol 5-phosphate to phosphatidylinositol 4,5-bis-phosphate. To understand the physiological role of these kinases, we generated mice that do not express phosphatidylinositol 5-phosphate 4-kinase β. These mice are hypersensitive to insulin and have reduced body weights compared to wild-type littermates. While adult male mice lacking phosphatidylinositol 5-phosphate 4-kinase β have significantly less body fat than wild-type littermates, female mice lacking phosphatidylinositol 5-phosphate 4-kinase β have increased insulin sensitivity in the presence of normal adiposity. Furthermore, in vivo insulin-induced activation of the protein kinase Akt is enhanced in skeletal muscle and liver from mice lacking phosphatidylinositol 5-phosphate 4-kinase β. These results indicate that phosphatidylinositol 5-phosphate 4-kinase β plays a role in determining insulin sensitivity and adiposity in vivo and suggest that inhibitors of this enzyme may be useful in the treatment of type 2 diabetes.
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Arai, N., H. Masuzaki, T. Tanaka, T. Ishii, S. Yasue, N. Kobayashi, T. Tomita, et al. "Ceramide and Adenosine 5′-Monophosphate-Activated Protein Kinase Are Two Novel Regulators of 11β-Hydroxysteroid Dehydrogenase Type 1 Expression and Activity in Cultured Preadipocytes." Endocrinology 148, no. 11 (November 1, 2007): 5268–77. http://dx.doi.org/10.1210/en.2007-0349.
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Increased activity of intracellular glucocorticoid reactivating enzyme, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in obese adipose tissue contributes to adipose dysfunction. As recent studies have highlighted a potential role of preadipocytes in adipose dysfunction, we tested the hypothesis that a variety of metabolic stress mediated by ceramide or AMP-activated protein kinase (AMPK) would regulate 11β-HSD1 in preadipocytes. The present study is the first to show that 1) expression of 11β-HSD1 in 3T3-L1 preadipocytes was robustly induced when cells were treated with cell-permeable ceramide analogue C2 ceramide, bacterial sphingomyelinase, and sphingosine 1-phosphate, 2) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced activation of AMPK augmented the expression and enzyme activity of 11β-HSD1, and 3) these results were reproduced in human preadipocytes. We demonstrate for the first time that C2 ceramide and AICAR markedly induced the expression of CCAAT/enhancer-binding protein (C/EBP) β and its binding to 11β-HSD1 promoter. Transient knockdown of C/EBPβ protein by small interfering RNA markedly attenuated the expression of 11β-HSD1 induced by C2 ceramide or AICAR. The present study provides novel evidence that ceramide- and AMPK-mediated signaling pathways augment the expression and activity of 11β-HSD1 in preadipocytes by way of C/EBPβ, thereby highlighting a novel, metabolic stress-related regulation of 11β-HSD1 in a cell-specific manner.
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Nishikawa, Kiyotaka, Alex Toker, Karen Wong, Paola A. Marignani, Franz-Josef Johannes, and Lewis C. Cantley. "Association of Protein Kinase Cμ with Type II Phosphatidylinositol 4-Kinase and Type I Phosphatidylinositol-4-phosphate 5-Kinase." Journal of Biological Chemistry 273, no. 36 (September 4, 1998): 23126–33. http://dx.doi.org/10.1074/jbc.273.36.23126.
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Luo, Jian-Dong, Tai-Ping Hu, Li Wang, Min-Sheng Chen, Shi-Ming Liu, and Alex F. Chen. "Sonic hedgehog improves delayed wound healing via enhancing cutaneous nitric oxide function in diabetes." American Journal of Physiology-Endocrinology and Metabolism 297, no. 2 (August 2009): E525—E531. http://dx.doi.org/10.1152/ajpendo.00308.2009.
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Sonic hedgehog (SHH) plays an important role in postnatal tissue repair. The present study tested the hypothesis that impaired SHH pathway results in delayed wound healing by suppressing cutaneous nitric oxide (NO) function in type 1 diabetes. Adult male C57/B6 mice and streptozotocin (STZ)-induced type 1 diabetic mice were used. Although cutaneous SHH and Patched-1 (Ptc-1 encoded by PTCH, PTCH 1) proteins were increased significantly on day 4 after wounding compared with day 0 in normal mice, both were decreased significantly in STZ-induced diabetic mice. Topical application of SHH restored wound healing delay in STZ-induced diabetic mice, with a concomitant augmentation of both cutaneous constitutive nitric oxide synthase (NOS) activity and nitrite level. The effects of SHH on wound healing and cutaneous NO function were markedly inhibited by SHH receptor inhibitor cyclopamine. After 24-h treatment in vitro, SHH (5–20 μg/ml) significantly increased cutaneous endothelial NOS protein expression, NOS activity and NO level in normal mice and STZ-induced diabetic mice in a concentration-dependent manner, an effect that was blunted by cyclopamine and NOS inhibitor Nω-nitro-l-arginine methyl ester. The phosphatidylinositol 3-kinase inhibitor LY-294002 significantly blunted the increase of NOS activity and NO level induced by SHH treatment in human umbilican vein endothelial cells. These results demonstrate that the SHH pathway is activated in a normal wound, and its reduction results in impaired NO function and wound healing in diabetes. Strategies aimed at augmenting the endogenous SHH pathway may provide an effective means in ameliorating delayed diabetic wound healing.
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Zhou, Ming-Sheng, Ivonne Hernandez Schulman, and Leopoldo Raij. "Role of angiotensin II and oxidative stress in vascular insulin resistance linked to hypertension." American Journal of Physiology-Heart and Circulatory Physiology 296, no. 3 (March 2009): H833—H839. http://dx.doi.org/10.1152/ajpheart.01096.2008.
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Insulin activation of the phosphatidylinositol 3-kinase (PI3K) pathway stimulates glucose uptake in peripheral tissues and synthesis of nitric oxide (NO) in the endothelium. Insulin resistance (IR) and hypertension frequently coexist, particularly among individuals with salt-sensitive hypertension. The mechanisms underlying this association are poorly understood. We investigated these mechanisms in a model of salt-sensitive hypertension in which we have previously shown that endothelial dysfunction is mediated by superoxide anion (O2−) linked to local ANG II. Dahl salt-sensitive rats were fed, for 6 wk, a normal salt diet (NS; 0.5% NaCl), high-salt diet (HS; 4% NaCl), HS plus the ANG II type 1 receptor (AT1R) blocker (ARB) candesartan (10 mg·kg−1·day−1), or HS plus the antioxidant tempol (172 mg/l in drinking water). Hypertensive (mean arterial pressure: 145 ± 4 vs. 102 ± 5 mmHg in NS, P < 0.05) rats manifested increased aortic AT1R mRNA (210%) and protein (101%) expression and O2− production (104%) and impaired endothelium-dependent relaxation (EDR) to acetylcholine [maximal response ( Emax): 68 ± 9 vs. 91 ± 8% in NS, P < 0.05]. ARB or tempol normalized O2− and EDR despite that they did not normalize mean arterial pressure, which was reduced only 25%. Hypertensive rats manifested metabolic IR (36% reduction in the glucose infusion rate by insulin clamp), impaired NO-mediated insulin-induced EDR ( Emax: 12 ± 5 vs. 32 ± 4% in NS, P < 0.05), and impaired insulin activation of PI3K/endothelial NO synthase. ARB or tempol improved insulin-mediated EDR, PI3K, Akt/ endothelial NO synthase phosphorylation, and metabolic IR (all P < 0.05). This study provides insight into the mechanisms that underlie the association between metabolic and hypertensive cardiovascular diseases and support the notion that O2− overproduction linked to tissue ANG II interferes with shared insulin signaling pathways in metabolic and cardiovascular tissues.
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Huang, Z., X. Huang, Y. Huang, Z. Li, Q. Huang, and T. Li. "POS0044 T315 SUPPRESSES OSTEOGENIC DIFFERENTIATION IN SAOS-2 CELLS BY INHIBITING PHOSPHORYLATION OF AKT." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 227.1–227. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2428.
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Background:New bone formation is common in the late stage of various inflammatory arthritis, while osteoblasts play a vital role in this process. Activation of PI3K/ Akt pathway promotes the differentiation and enhances the function of osteoblasts [1]. T315 is a novel small molecule drug, which may induce apoptosis and suppress the expression of cellular markers of chronic lymphocytic leukemia cells by disrupting PI3K/ Akt pathway [2]. However, the lack of study focuses on the influence of T31T on the other cells, except tumor cell lines.Objectives:We aimed to assess the effect of T315 on human osteoblast-like Saos-2 cells, while its potential mechanism in PI3K/ Akt pathway was evaluated as well.Methods:(1) Saos-2 was stimulated with an osteogenic reagent which contained L-ascorbic acid, β-glycerophosphoric acid, and dexamethasone. The concentration of T315 was adjusted to 0μg/ml, 1μg/ml, and 2μg/ml in the culture medium. (2) Alizarin red stain and alkaline phosphatase (ALP) stain were performed at d0, d7, d14, and d21 after being treated with T315. (3) Cellular protein was extracted at d0, d3, and d6 after being treated with T315, then ALP activity was tested based on a recommendation from the manufacturer of the kit. (4) Collagen type 1α2 Chain (Col1α2) and osteocalcin (OCN), two osteogenic markers, were measured through western blot, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. (5) Phospho-phosphoinositide 3-kinase (pPI3K), phospho-protein kinase B (pAkt), and runt-related transcription factor 2 (Runx2) was tested through western blot as well. GAPDH or protein kinase (Akt) was chosen as an internal reference as appropriate. (6) Analysis of variance with the least significant difference was used to analyze the data. A P<0.05 was considered statistically significant.Results:(1) The higher concentration of T315 related to the less relative mineralized area and the positive area of ALP, while longer incubation time with T315 decreased these regions as well (Figure 1A-C). (2) T315 reduced the activity of ALP accordingly (Figure 1D). (3) T315 suppressed the protein expression of Col1α2 and OCN in a dose-dependent and time-dependent manner (Figure 1E, F). (4) T315 did not alter pPI3K, but it inhibited the phosphorylation of Akt (Figure 1G, H). (5) Runx2 was reduced because of the greater dose or longer incubation time with T315 (Figure 1).Conclusion:T315 inhibits the differentiation of osteoblasts through inhibiting the phosphorylation of Akt. Surprisingly, pPI3K seldom changes in this process, so its detail mechanism should be investigated in further.References:[1]Exp Biol Med (Maywood) 2020;245(6):552-561.[2]Blood 2015;125(2):284-295.Figure 1.Effect of T315 on Saos-2 cells and PI3K/Akt pathway. (A) Alizarin red stain and ALP stain at d21. (B) Relative mineralized area in Alizarin red stain. (C) Positive area in ALP stain. (D) ALP activity. Western blot analysis and its bands at d6 for Col1α2 (E), OCN (F), pPI3K (G), pAkt (H), and Runx2 (I). Results were normalized by GAPDH or Akt. ALP: Alkaline phosphatase; Col1α2: Collagen type 1α2 Chain; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; OCN: Osteocalcin; pPI3K: Phospho-phosphoinositide 3-kinase; pAkt: Phospho-protein kinase B; Akt: Protein kinase; Runx2: Runt-related transcription factor 2. a Compared with d3 in the same concentration of T315, P<0.05. b Compared with d0 in the same concentration of T315, P<0.05. c Compared with 1μg/ml T315 in the same incubation time, P<0.05. d Compared with 2μg/ml T315 in the same incubation time, P<0.05.Disclosure of Interests:None declared.