title: Reduced insulin receptor signaling in retinal Müller cells cultured in high glucose.
abstract: OBJECTIVE
  To measure key proteins involved in insulin resistance in retinal Müller cells.
  
  
  METHODS
  Cells known as retinal Müller cells were cultured in normal (5 mM) or high glucose (25 mM) to mimic a diabetic condition. Cells were treated with 50 nM Compound 49b, a novel β-adrenergic receptor agonist. Additional cells were treated with small interfering RNA (siRNA) against protein kinase A or cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB). Western blotting or enzyme-linked immunosorbent assay (ELISA) measurements were made for protein changes in TNFα, suppressor of cytokine signaling 3, insulin receptor substrate 1 (IRS-1), insulin receptor (IR), Akt, and cell death proteins (Fas, fas ligand, cytochrome C, Bax, cleaved caspase 3, and Bcl-xL).
  
  
  RESULTS
  Hyperglycemia significantly increased TNFα and suppressor of cytokine signaling 3 levels. This was associated with increased phosphorylation of IRS-1(Ser307) and IR(Tyr960), with decreased phosphorylation of IR(Tyr1150/1151) and Akt(Ser473). The reduced insulin receptor and Akt phosphorylation led to a significant increase in proapoptotic proteins. Compound 49b reversed the loss of Akt and IR(Tyr1150/1151) phosphorylation, reducing Müller cell apoptosis.
  
  
  CONCLUSIONS
  Hyperglycemia-induced TNFα levels promote insulin resistance in retinal Müller cells, noted through increased phosphorylation of IRS-1(Ser307) and IR(Tyr960). The dysfunctional insulin signaling increases apoptosis of retinal Müller cells, which is blocked through treatment with Compound 49b. Taken together, β-adrenergic receptor agonists may protect retinal Müller cells through maintenance of normal insulin receptor signaling.
authors.affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
authors.forename: Youde
authors.initials: Y
authors.lastname: Jiang
authors.forename: Jayaprakash
authors.initials: J
authors.lastname: Pagadala
authors.forename: Duane
authors.initials: D
authors.lastname: Miller
authors.forename: Jena J
authors.initials: JJ
authors.lastname: Steinle
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D012160:Retina / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D055504:Insulin Receptor Substrate Proteins; D018972:Insulin-Like Growth Factor Binding Protein 3; C491410:Socs3 protein, rat; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014409:Tumor Necrosis Factor-alpha; D011972:Receptor, Insulin; D005947:Glucose