3951f7d668e8470458e6eb1fad1bae7c pmc_34884763.pdf 1984d88b88f94eeb35f3eaad2a776609abab951d pmc_34884763.pdf 5b8a38a2d12b8f9abae5117af7fb012fbf75310ef43efb7405de347f3706bb43 pmc_34884763.pdf Title: Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles Subject: The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy. Keywords: insulin resistance; neuregulin 4; adipocytes; autophagy; inflammation; insulin receptor; Glut4 Author: Francisco Díaz-Sáez, Carla Blanco-Sinfreu, Adrià Archilla-Ortega, David Sebastian, Montserrat Romero, Maria Isabel Hernández-Alvarez, Sílvia Mora, Xavier Testar, Wifredo Ricart, José Manuel Fernández-Real, José María Moreno-Navarrete, Julián Aragonés, Marta Camps, Antonio Zorzano and Anna Gumà Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Nov 30 11:12:31 2021 CET ModDate: Tue Nov 30 11:18:19 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5697595 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GSWAKP+VnURWPalladioL Type 1 Custom yes yes no 10 0 YLCAFF+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 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DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Nrg4 Deficiency Has No Effect on Differentiation of 3T3-L1 PRE-Adipocytes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Nrg4-Deficient Adipocytes Have Impaired Insulin-Stimulated Glucose Transport Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Nrg4 KD Adipocytes Show Enhanced Inflammation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Treatment of Nrg4 KD Adipocytes with Conditioned Medium from Control Adipocytes or with Recombinant Nrg4 Reverses Inflammation and Restores Insulin Receptor and Glut4 Content Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Sodium Salicylate and Dexamethasone Restore the Expression of the Insulin Receptor, but Not the Content of Glut4 Protein in Nrg4 KD Adipocytes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Autophagy Is Enhanced in Nrg4 KD Adipocytes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Blockade of Autophagy Restores Glut4 and GSV Proteins Content in Nrg4 KD Adipocytes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: Reagents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: White Adipose Tissue Fractionation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: T3-L1 Adipocyte Cell Culture and Differentiation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Lentiviral Production and Infection of 3T3-L1 Preadipocytes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Cell Treatments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Measurement of 2-d-Deoxyglucose Uptake Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: RNA Extraction and Quantitative PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Protein Extraction and Western Blotting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Info: Title: Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles Author: Francisco Díaz-Sáez, Carla Blanco-Sinfreu, Adrià Archilla-Ortega, David Sebastian, Montserrat Romero, Maria Isabel Hernández-Alvarez, Sílvia Mora, Xavier Testar, Wifredo Ricart, José Manuel Fernández-Real, José María Moreno-Navarrete, Julián Aragonés, Marta Camps, Antonio Zorzano and Anna Gumà Subject: The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy. Keywords: insulin resistance; neuregulin 4; adipocytes; autophagy; inflammation; insulin receptor; Glut4 Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Nov 30 11:12:31 CET 2021 ModDate: Tue Nov 30 11:18:19 CET 2021 ID: 0xd30a63091e8a014a9311511bb4f9ddf6, 0xd30a63091e8a014a9311511bb4f9ddf6 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: KESBIZ+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: KESBIZ+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: SOFIBE+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: SOFIBE+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: GPUOQI+URWPalladioL-Roma 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