title: Acute knockdown of the insulin receptor or its substrates Irs1 and 2 in 3T3-L1 adipocytes suppresses adiponectin production.
abstract: Loss of function of the insulin receptor (INSR) in humans produces severe insulin resistance. Unlike "common" insulin resistance, this is associated with elevated plasma levels of the insulin-sensitising, adipose-derived protein adiponectin. The underlying mechanism for this paradox is unclear, and it is at odds with the acute stimulation of adiponectin secretion reported on insulin treatment of cultured adipocytes. Given recent evidence for ligand-independent actions of the INSR, we used a lentiviral system to knock down Insr or its substrates Irs1 and Irs2 conditionally in 3T3-L1 murine preadipocytes/adipocytes to assess whether acute loss of their expression has different consequences to withdrawal of insulin. Efficient knockdown of either Insr or Irs1/2 was achieved by conditional shRNA expression, severely attenuating insulin-stimulated AKT phosphorylation and glucose uptake. Dual knockdown of Irs1 and Irs2 but not Insr in preadipocytes impaired differentiation to adipocytes. Acute knockdown of Insr or both Irs1 and Irs2 in adipocytes increased Adipoq mRNA expression but reduced adiponectin secretion, assessed by immunoassay. Knockdown sustained for 14 days also reduced immunoassay-detected adiponectin secretion, and moreover induced delipidation of the cells. These findings argue against a distinct effect of Insr deficiency to promote adiponectin secretion as the explanation for paradoxical insulin receptoropathy-related hyperadiponectinaemia.
authors.affiliation: The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK.|The National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, UK.
authors.forename: Matthijs P
authors.initials: MP
authors.lastname: Groeneveld
authors.affiliation: The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK.|The National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, UK.
authors.forename: Gemma V
authors.initials: GV
authors.lastname: Brierley
authors.affiliation: The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK.|The National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, UK.
authors.forename: Nuno M
authors.initials: NM
authors.lastname: Rocha
authors.affiliation: The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK.|The National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, UK.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Siddle
authors.affiliation: The University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Cambridge, UK.|The National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, UK.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Semple
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D052242:Adiponectin / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism
chemical_list: D052242:Adiponectin; C499919:Adipoq protein, mouse; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse; D011972:Receptor, Insulin