title: Insulin activates hypoxia-inducible factor-1alpha in human and rat vascular smooth muscle cells via phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathways: impairment in insulin resistance owing to defects in insulin signalling.
abstract: OBJECTIVE
  We previously demonstrated that insulin stimulates vascular endothelial growth factor (VEGF) synthesis and secretion via phosphatidylinositol-3 kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways in vascular smooth muscle cells (VSMC) from humans and from insulin-sensitive lean Zucker fa/+ rats. We also showed that this effect is attenuated in VSMC from insulin-resistant obese Zucker fa/fa rats. As it is not known whether the effects of insulin on VEGF involve activation of hypoxia-inducible factor-1 (HIF-1), we aimed to evaluate: (1) whether insulin modulates HIF-1alpha protein synthesis and activity; (2) the insulin signalling pathways involved; and (3) the role of insulin resistance.
  
  
  METHODS
  Using aortic VSMC taken from humans and Zucker rats and cultured in normoxia, the following were evaluated: (1) dose-dependent (0.5, 1, 2 nmol/l) and time-dependent (2, 4, 6 h) effects exerted by insulin on HIF-1alpha content in both nucleus and cytosol, measured by Western blots; (2) insulin effects on HIF-1 DNA-binding activity on the VEGF gene, measured by electrophoretic mobility shift assay; and (3) involvement of the insulin signalling molecules in these insulin actions, by using the following inhibitors: LY294002 (PI3-K), PD98059 (extracellular signal regulated kinase [ERK]), SP600125 (Jun N terminal kinase [JNK]), SB203580 (p38 mitogen-activated protein kinase) and rapamycin (mammalian target of rapamycin), and by detecting the insulin signalling molecules by Western blots.
  
  
  RESULTS
  In aortic VSMC from humans and Zucker fa/+ rats cultured in normoxia insulin increases the HIF-1alpha content in cytosol and nucleus via dose- and time-dependent mechanisms, and HIF-1 DNA-binding activity on the VEGF gene. The insulin-induced increase of HIF-1alpha is blunted by the translation inhibitor cycloheximide, LY294002, PD98059, SP600125 and rapamycin, but not by SB203580. It is also reduced in Zucker fa/fa rats, which present an impaired ability of insulin to induce Akt, ERK-1/2 and JNK-1/2 phosphorylation.
  
  
  CONCLUSIONS
  These results provide a biological mechanism for the impaired collateral vessel formation in obesity.
authors.affiliation: Diabetes Unit, Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, I-10043, Orbassano, Turin, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Doronzo
authors.forename: I
authors.initials: I
authors.lastname: Russo
authors.forename: L
authors.initials: L
authors.lastname: Mattiello
authors.forename: C
authors.initials: C
authors.lastname: Riganti
authors.forename: G
authors.initials: G
authors.lastname: Anfossi
authors.forename: M
authors.initials: M
authors.lastname: Trovati
mesh_terms: D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology* / Q000502:physiology*; D007333:Insulin Resistance / Q000502:physiology*; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D011924:Rats, Zucker; D015398:Signal Transduction / Q000502:physiology
chemical_list: D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007093:Imidazoles; D007328:Insulin; D011725:Pyridines; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases; C093642:SB 203580