title: Short-term high glucose exposure impairs insulin signaling in endothelial cells.
abstract: BACKGROUND
  Hyperglycemia is the hallmark of diabetes and its cardiovascular complications. Insulin plays an important role in the regulation of vascular homeostasis and maintenance of endothelial function. Insulin signaling occurs after binding to the insulin receptor, causing activation of two separate and parallel pathways: PI3K/AKT/eNOS and Ras/Raf/MAPK pathways. AKT phosphorylates eNOS at Ser1177, resulting in increased nitric oxide production and vasodilation. The MAPK pathway results in endothelin-1 production and vasoconstriction and mitogenic effects.
  
  
  METHODS
  We studied the effects of physiological insulin treatment in human umbilical vein endothelial cells (HUVECs) on the two pathways under high glucose conditions, which mimic the in vivo condition of hyperglycemia. HUVECs were incubated with insulin at different physiological concentrations (from 10(-10) to 10(-8) M) for 30 min after 24 h of exposition to normal (5 mmol/L, NG) or high glucose (25 mmol/L, HG). Phosphorylated forms of AKT, eNOS, ERK1/2, p38, JNK and insulin receptor-β subunit (IRβ) were evaluated.
  
  
  RESULTS
  In normal glucose, the active phosphorylated forms of AKT, eNOS, ERK1/2, p38 and JNK were increased in insulin treated cells, in a dose-dependent manner. In high glucose, insulin was not able to activate the PI3K/AKT/eNOS pathway, with the phosphorylated form of eNOS reduced with respect to the control. However, insulin was able to induce the up-regulation of phospho-ERK1/2, -p38 and -JNK. Moreover, we found reduced levels of IRβ phosphorylated form in high glucose as compared to the control. Insulin was able to increase phospho-IRβ in normal glucose but not in high glucose, in which the total protein levels remained reduced.
  
  
  CONCLUSIONS
  Exposure to short-term high glucose negatively affects insulin signaling even when physiological insulin concentrations are added. The impairment of the PI3K/AKT/eNOS pathway after physiological insulin treatment could contribute to detrimental effects on cardiovascular homeostasis under high glucose conditions, and might shift toward the activation of certain mitogenic effectors, such as ERK1/2, p38 and JNK, the only ones that respond to physiological insulin treatment in high glucose.
authors: Insititut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Hospital Clinic, C/Rosselló, 149-153, 08036, Barcelona, Spain. vnigris@clinic.ub.es.
authors: Valeria
authors: V
authors: De Nigris
authors: Insititut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Hospital Clinic, C/Rosselló, 149-153, 08036, Barcelona, Spain. gpujadas@clinic.ub.es.
authors: Gemma
authors: G
authors: Pujadas
authors: Insititut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Hospital Clinic, C/Rosselló, 149-153, 08036, Barcelona, Spain. lulasala@clinic.ub.es.
authors: Lucia
authors: L
authors: La Sala
authors: Experimental Models in Clinical Pathology, INRCA-IRCCS National Institute, Ancona, Italy. r.testa@inrca.it.
authors: Roberto
authors: R
authors: Testa
authors: Department of Cardiovascular and Metabolic Diseases, IRCCS Gruppo Multimedica, Sesto San Giovanni, MI, Italy. stefano.genovese@multimedica.it.
authors: Stefano
authors: S
authors: Genovese
authors: Insititut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Hospital Clinic, C/Rosselló, 149-153, 08036, Barcelona, Spain. aceriell@clinic.ub.es.
authors: Antonio
authors: A
authors: Ceriello
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011972:Receptor, Insulin / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors
chemical_list: D015703:Antigens, CD; D007328:Insulin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C435941:INSR protein, human; D011972:Receptor, Insulin; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose