| 1. |
- Borgegard, Tomas, et al.
(författare)
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Alzheimers Disease: Presenilin 2-Sparing gamma-Secretase Inhibition Is a Tolerable A beta Peptide-Lowering Strategy
- 2012
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Ingår i: Journal of Neuroscience. - : Society for Neuroscience. - 0270-6474 .- 1529-2401. ; 32:48, s. 17297-17305
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Tidskriftsartikel (refereegranskat)abstract
- gamma-Secretase inhibition represents a major therapeutic strategy for lowering amyloid beta (A beta) peptide production in Alzheimers disease (AD). Progress toward clinical use of gamma-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The gamma-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between A beta production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1(PS1) over PS2 subclass of gamma-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain A beta levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious gamma-secretase targeting strategy for AD.
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| 2. |
- Borgegård, Tomas, et al.
(författare)
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Alzheimer's Disease : Presenilin 2-Sparing γ-Secretase Inhibition Is a Tolerable Aβ Peptide-Lowering Strategy
- 2012
-
Ingår i: Journal of Neuroscience. - 0270-6474 .- 1529-2401. ; 32:48, s. 17297-17305
-
Tidskriftsartikel (refereegranskat)abstract
- γ-Secretase inhibition represents a major therapeutic strategy for lowering amyloid β (Aβ) peptide production in Alzheimer's disease (AD). Progress toward clinical use of γ-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The γ-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between Aβ production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1 (PS1) over PS2 subclass of γ-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain Aβ levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious γ-secretase targeting strategy for AD.
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| 3. |
- Dyczynski, Matheus, et al.
(författare)
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Targeting autophagy by small molecule inhibitors of vacuolar protein sorting 34 (Vps34) improves the sensitivity of breast cancer cells to Sunitinib
- 2018
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Ingår i: Cancer Letters. - : Elsevier. - 0304-3835 .- 1872-7980. ; 435, s. 32-43
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to chemotherapy is a challenging problem for treatment of cancer patients and autophagy has been shown to mediate development of resistance. In this study we systematically screened a library of 306 known anti-cancer drugs for their ability to induce autophagy using a cell-based assay. 114 of the drugs were classified as autophagy inducers; for 16 drugs, the cytotoxicity was potentiated by siRNA-mediated knock-down of Atg7 and Vps34. These drugs were further evaluated in breast cancer cell lines for autophagy induction, and two tyrosine kinase inhibitors, Sunitinib and Erlotinib, were selected for further studies. For the pharmacological inhibition of autophagy, we have characterized here a novel highly potent selective inhibitor of Vps34, SB02024. SB02024 blocked autophagy in vitro and reduced xenograft growth of two breast cancer cell lines, MDA-MB-231 and MCF-7, in vivo. Vps34 inhibitor significantly potentiated cytotoxicity of Sunitinib and Erlotinib in MCF-7 and MDA-MB-231 in vitro in monolayer cultures and when grown as multicellular spheroids. Our data suggests that inhibition of autophagy significantly improves sensitivity to Sunitinib and Erlotinib and that Vps34 is a promising therapeutic target for combination strategies in breast cancer.
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| 4. |
- Gustavsson, Johanna, 1956-, et al.
(författare)
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Localization of the insulin receptor in caveolae of adipocyte plasma membrane
- 1999
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Ingår i: The FASEB Journal. - 0892-6638 .- 1530-6860. ; 13:14, s. 1961-1971
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Tidskriftsartikel (refereegranskat)abstract
- The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with β-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. β-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.
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| 5. |
- Karlsson, Margareta, 1942-, et al.
(författare)
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Insulin induces translocation of glucose transporter GLUT4 to plasma membrane caveolae in adipocytes
- 2002
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 16:2, s. 249-251
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Tidskriftsartikel (refereegranskat)abstract
- Insulin-stimulated glucose uptake in muscle and adipose tissue is the result of translocation of insulin-regulated glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane. Here we report that GLUT4 in the plasma membrane of 3T3-L1 adipocytes were located predominantly in caveolae invaginations: by immunogold electron microscopy of plasma membranes, 88% of GLUT4 were localized to caveolae structures and this distribution within the plasma membrane was not affected by insulin. By immunofluorescence microscopy, a major part of GLUT 4 was colocalized with caveolin. The total amount of GLUT4 in the plasma membrane increased 2.2-fold in response to insulin as determined by immunogold electron or immunofluorescence microscopy. GLUT4 were enriched in caveolae fractions isolated without detergents from plasma membranes of rat adipocytes. In these fractions, GLUT4 were largely confined to caveolin-containing membranes of the caveolae preparation isolated from insulin-stimulated cells, determined by electron microscopy. Insulin increased the amount of GLUT4 2.7-fold in this caveolae fraction. Caveolae were purified further by immunoisolation with antibodies against caveolin. The amount of GLUT4 increased to the same extent in the immunopurified caveolae as in the cruder caveolae fractions from insulin-stimulated cells. We conclude that insulin induces translocation of GLUT4 to caveolae.
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| 6. |
- Parpal, Santiago, et al.
(författare)
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Cholesterol Depletion Disrupts Caveolae and Insulin Receptor Signaling for Metabolic Control via Insulin Receptor Substrate-1, but Not for Mitogen-activated Protein Kinase Control
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:13, s. 9670-9678
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Tidskriftsartikel (refereegranskat)abstract
- Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Strålfors, P. (1999) FASEB. J. 13, 1961–1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with β-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by 125I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects.
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| 7. |
- Parpal, Santiago
(författare)
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Mechanisms of insulin signaling and the role of caveolae
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Insulin regulates metabolic as well as mitogenic processes in target cells, involving a large number of mediators of signal transduction. In its role as a growth factor, insulin stimulates cell growth, in a process we demonstrate requires the participation of the Raf-1 kinase.Caveolae are invaginations of the plasma membrane, involved in signal transduction and intracellular transport of cholesterol. Caveolae are enriched in cholesterol, sphingolipids and the constituent protein caveolin. Herein we report that the insulin receptor is located in caveolae of plasma membrane from adipocytes. By confocal and electron microscopy we show co-localization of caveolin and the insulin receptor. Additionally, the insulin receptor independently of insulin stimulation is enriched in caveolae isolated by cell fractionation.Cholesterol depletion has been shown to flatten caveolae and affect processes which occur in these domains. We show that depletion of cholesterol in adipocytes destroys caveolae and inhibits insulin-stimulated tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1 ), without affecting insulin receptor ligand binding or its autophosphorylation. Cholesterol-depleted adipocytes showed a decreased insulin-stimulated glucose uptake and phosphorylation of A TP citrate-lyase. Cholesterol depletion did not affect insulin's effect on the MAPK kinases ERK 1/2. She, which has been described to mediate an alternative pathway to that mediated by IRS-1 for insulin mitogenic regulation, was not involved in the regulation of the MAP kinases by insulin in adipocytes. We conclude that some other mediators which are not dependent on caveolae integrity must exist for regulation of this pathway.The effects of cholesterol depletion on caveolae and insulin signaling prompted us to study caveolae in models of insulin resistance. We show that adipocytes from the obese and insulin resistant Zucker fa/fa rats have reduced amounts of cholesterol in caveolae compared with their lean littermates. Adipocytes of Zucker fa/fa rats have been shown to express high levels of TNF-α. We demonstrate that TNF-α treatment lowers the amount of cholesterol in caveolae in adipocytes from normal rats.The results presented in this thesis demonstrate that insulin signaling originates in caveolae invaginations of the plasma membrane where the insulin receptor is located. Caveolae are required for certain metabolic effects of insulin but not for activation of the MAP kinase pathway, a scenario similar to what is found in cases of insulin resistance and type 2 diabetes. Moreover, alteration of the amount of cholesterol or caveolae leads to insulin resistance, suggesting that caveolae play a central role in insulin resistance and diabetes.
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| 8. |
- Parpal, Santiago, et al.
(författare)
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Reduced content of cholesterol in caveolae of adipocytes from insulin resistant, obese, and diabetic Zucker fa/fa rats
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The Zucker fa/fa rat has a mutation in the leptin receptor and therefore over-eats and becomes grossly obese. The animal develops insulin resistance and tumor necrosis factor a (TNFa) has been implicated in the pathogenesis of the insulin resistance that thls animal model of type 2 diabetes exhibits. We have shown that the insulin receptor is located in caveolae of the plasma membrane and that reduction of caveolar cholesterol content makes adipocytes insulin resistant (Parpal et a., J. Biol. Chem. 276 (2001)9670-9678). Here we show that adipocytes from the Zucker fa/fa rat are insulin resistant and that this can be explained by a 50 % lower content of cholesterol in the caveolae compared to lean rats. Moreover, TNFa treatment reduces the caveolar content of cholesterol in normal rat adipocytes. An enhanced production of TNFa may thus reduce caveolar cholesterol levels and make the Zucker fa/fa rat insulin resistant.
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| 9. |
- Törnkvist, Åsa, et al.
(författare)
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Inhibition of Raf-1 kinase expression abolishes insulin stimulation of DNA synthesis in H4IIE hepatoma cells
- 1994
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 269:19, s. 13919-13921
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Tidskriftsartikel (refereegranskat)abstract
- The involvement of Raf-1 kinase in the insulin signal transduction chain leading to control of cell proliferation was studied in the H4IIE rat hepatoma cell line by inhibiting expression of the kinase with antisense oligodeoxyribonucleotide directed against Raf-1 mRNA. Antisense oligonucleotide was found to reduce (at 2 microM) or completely block (at 15 microM) the stimulation by insulin of DNA synthesis, measured as thymidine incorporation. The residual DNA synthesis seen in the absence of insulin stimulation was also inhibited by the Raf-1 kinase antisense oligonucleotide.
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| 10. |
- Wanngren, Johanna, et al.
(författare)
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Second generation γ-secretase modulators exhibit different modulation of Notch β and Aβ production.
- 2012
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 287:39, s. 32640-50
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Tidskriftsartikel (refereegranskat)abstract
- The γ-secretase complex is an appealing drug target when the therapeutic strategy is to alter amyloid-β peptide (Aβ) aggregation in Alzheimer disease. γ-Secretase is directly involved in Aβ formation and determines the pathogenic potential of Aβ by generating the aggregation-prone Aβ42 peptide. Because γ-secretase mediates cleavage of many substrates involved in cell signaling, such as the Notch receptor, it is crucial to sustain these pathways while altering the Aβ secretion. A way of avoiding interference with the physiological function of γ-secretase is to use γ-secretase modulators (GSMs) instead of inhibitors of the enzyme. GSMs modify the Aβ formation from producing the amyloid-prone Aβ42 variant to shorter and less amyloidogenic Aβ species. The modes of action of GSMs are not fully understood, and even though the pharmacology of GSMs has been thoroughly studied regarding Aβ generation, knowledge is lacking about their effects on other substrates, such as Notch. Here, using immunoprecipitation followed by MALDI-TOF MS analysis, we found that two novel, second generation GSMs modulate both Notch β and Aβ production. Moreover, by correlating S3-specific Val-1744 cleavage of Notch intracellular domain (Notch intracellular domain) to total Notch intracellular domain levels using immunocytochemistry, we also demonstrated that Notch intracellular domain is not modulated by the compounds. Interestingly, two well characterized, nonsteroidal anti-inflammatory drugs (nonsteroidal anti-inflammatory drug), R-flurbiprofen and sulindac sulfide, affect only Aβ and not Notch β formation, indicating that second generation GSMs and nonsteroidal anti-inflammatory drug-based GSMs have different modes of action regarding Notch processing.
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