Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta
IntroductionLipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and p...
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Frontiers Media S.A.
2023-02-01
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| Series: | Frontiers in Endocrinology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fendo.2023.1092024/full |
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| author | Natascha Berger Tom van der Wel Birgit Hirschmugl Birgit Hirschmugl Thomas Baernthaler Juergen Gindlhuber Juergen Gindlhuber Nermeen Fawzy Thomas Eichmann Thomas Eichmann Ruth Birner-Gruenberger Ruth Birner-Gruenberger Robert Zimmermann Robert Zimmermann Mario van der Stelt Christian Wadsack Christian Wadsack |
| author_facet | Natascha Berger Tom van der Wel Birgit Hirschmugl Birgit Hirschmugl Thomas Baernthaler Juergen Gindlhuber Juergen Gindlhuber Nermeen Fawzy Thomas Eichmann Thomas Eichmann Ruth Birner-Gruenberger Ruth Birner-Gruenberger Robert Zimmermann Robert Zimmermann Mario van der Stelt Christian Wadsack Christian Wadsack |
| author_sort | Natascha Berger |
| collection | DOAJ |
| description | IntroductionLipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase α and β (DAGLα, DAGLβ) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks.MethodsDAGLα and DAGLβ mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGLβ transcripts to different cell types of the placenta. DAGLβ activity was determined by in- gel and MS-based activity-based protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek™ lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 µM] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined.ResultsWe demonstrate that mRNA expression of DAGLβ prevails in placental tissue, compared to DAGLα (p ≤ 0.0001) and that DAGLβ is mainly located to CK7 positive trophoblasts (p ≤ 0.0001). Although few DAGLα transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLβ is the principal DAGL in the placenta. DAGLβ dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLβ by DH376 led to reduced MAG tissue levels (p ≤ 0.01), including 2-AG (p≤0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta.DiscussionOur results emphasize the role of DAGLβ activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies. |
| first_indexed | 2024-04-10T15:19:19Z |
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| institution | Directory Open Access Journal |
| issn | 1664-2392 |
| language | English |
| last_indexed | 2024-04-10T15:19:19Z |
| publishDate | 2023-02-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Endocrinology |
| spelling | doaj.art-554df234a0eb4e46ba913fb5910999172023-02-14T15:57:30ZengFrontiers Media S.A.Frontiers in Endocrinology1664-23922023-02-011410.3389/fendo.2023.10920241092024Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placentaNatascha Berger0Tom van der Wel1Birgit Hirschmugl2Birgit Hirschmugl3Thomas Baernthaler4Juergen Gindlhuber5Juergen Gindlhuber6Nermeen Fawzy7Thomas Eichmann8Thomas Eichmann9Ruth Birner-Gruenberger10Ruth Birner-Gruenberger11Robert Zimmermann12Robert Zimmermann13Mario van der Stelt14Christian Wadsack15Christian Wadsack16Department of Obstetrics and Gynecology, Medical University of Graz, Graz, AustriaDepartment of Molecular Physiology, Leiden Institute of Chemistry, Leiden University and Oncode Institute, Leiden, NetherlandsDepartment of Obstetrics and Gynecology, Medical University of Graz, Graz, AustriaBioTechMed-Graz, Graz, AustriaOtto Loewi Research Center, Division of Pharmacology, University of Graz, Graz, AustriaLudwig Boltzmann Institute for Lung Vascular Research, Graz, AustriaDiagnostic and Research Center of Molecular Medicine, Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, AustriaInstitute of Molecular Biosciences, University of Graz, Graz, AustriaBioTechMed-Graz, Graz, AustriaCore Facility Mass Spectrometry, Center for Medical Research (ZMF), Medical University of Graz, Graz, AustriaDiagnostic and Research Center of Molecular Medicine, Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, AustriaInstitute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, AustriaBioTechMed-Graz, Graz, AustriaInstitute of Molecular Biosciences, University of Graz, Graz, AustriaDepartment of Molecular Physiology, Leiden Institute of Chemistry, Leiden University and Oncode Institute, Leiden, NetherlandsDepartment of Obstetrics and Gynecology, Medical University of Graz, Graz, AustriaBioTechMed-Graz, Graz, AustriaIntroductionLipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase α and β (DAGLα, DAGLβ) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks.MethodsDAGLα and DAGLβ mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGLβ transcripts to different cell types of the placenta. DAGLβ activity was determined by in- gel and MS-based activity-based protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek™ lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 µM] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined.ResultsWe demonstrate that mRNA expression of DAGLβ prevails in placental tissue, compared to DAGLα (p ≤ 0.0001) and that DAGLβ is mainly located to CK7 positive trophoblasts (p ≤ 0.0001). Although few DAGLα transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLβ is the principal DAGL in the placenta. DAGLβ dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLβ by DH376 led to reduced MAG tissue levels (p ≤ 0.01), including 2-AG (p≤0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta.DiscussionOur results emphasize the role of DAGLβ activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies.https://www.frontiersin.org/articles/10.3389/fendo.2023.1092024/fullhuman placentalipid metabolismendocannabinoid system2-arachidonoylglyceroldiacylglycerol lipaseactivity-based protein profiling |
| spellingShingle | Natascha Berger Tom van der Wel Birgit Hirschmugl Birgit Hirschmugl Thomas Baernthaler Juergen Gindlhuber Juergen Gindlhuber Nermeen Fawzy Thomas Eichmann Thomas Eichmann Ruth Birner-Gruenberger Ruth Birner-Gruenberger Robert Zimmermann Robert Zimmermann Mario van der Stelt Christian Wadsack Christian Wadsack Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta Frontiers in Endocrinology human placenta lipid metabolism endocannabinoid system 2-arachidonoylglycerol diacylglycerol lipase activity-based protein profiling |
| title | Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta |
| title_full | Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta |
| title_fullStr | Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta |
| title_full_unstemmed | Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta |
| title_short | Inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta |
| title_sort | inhibition of diacylglycerol lipase β modulates lipid and endocannabinoid levels in the ex vivo human placenta |
| topic | human placenta lipid metabolism endocannabinoid system 2-arachidonoylglycerol diacylglycerol lipase activity-based protein profiling |
| url | https://www.frontiersin.org/articles/10.3389/fendo.2023.1092024/full |
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