Insulin activates hypoxia-inducible factor-1α 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

G. Doronzo, I. Russo, L. Mattiello, C. Riganti, G. Anfossi, M. Trovati

Research output: Contribution to journalArticle

Abstract

Aims/hypothesis: 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-1α 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-1α 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-1α 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-1α 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/interpretation: These results provide a biological mechanism for the impaired collateral vessel formation in obesity.

Original languageEnglish
Pages (from-to)1049-1063
Number of pages15
JournalDiabetologia
Volume49
Issue number5
DOIs
Publication statusPublished - May 2006

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Phosphatidylinositol 3-Kinase
Hypoxia-Inducible Factor 1
Mitogen-Activated Protein Kinases
Vascular Smooth Muscle
Smooth Muscle Myocytes
Insulin Resistance
Insulin
Zucker Rats
Vascular Endothelial Growth Factor A
Sirolimus
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Cytosol
Phosphotransferases
Western Blotting
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinase 1
DNA
Extracellular Signal-Regulated MAP Kinases
Electrophoretic Mobility Shift Assay
p38 Mitogen-Activated Protein Kinases

Keywords

  • c-Jun N-terminal kinase
  • Hypoxia-inducible factor
  • Insulin
  • Insulin resistance
  • Mitogen-activated protein kinase
  • Obesity
  • Phosphatidylinositol-3 kinase
  • Vascular endothelial growth factor
  • Vascular smooth muscle
  • Zucker rats

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Internal Medicine

Cite this

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title = "Insulin activates hypoxia-inducible factor-1α 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 = "Aims/hypothesis: 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-1α 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-1α 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-1α 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-1α 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/interpretation: These results provide a biological mechanism for the impaired collateral vessel formation in obesity.",
keywords = "c-Jun N-terminal kinase, Hypoxia-inducible factor, Insulin, Insulin resistance, Mitogen-activated protein kinase, Obesity, Phosphatidylinositol-3 kinase, Vascular endothelial growth factor, Vascular smooth muscle, Zucker rats",
author = "G. Doronzo and I. Russo and L. Mattiello and C. Riganti and G. Anfossi and M. Trovati",
year = "2006",
month = "5",
doi = "10.1007/s00125-006-0156-0",
language = "English",
volume = "49",
pages = "1049--1063",
journal = "Diabetologia",
issn = "0012-186X",
publisher = "Springer Verlag",
number = "5",

}

TY - JOUR

T1 - Insulin activates hypoxia-inducible factor-1α in human and rat vascular smooth muscle cells via phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathways

T2 - Impairment in insulin resistance owing to defects in insulin signalling

AU - Doronzo, G.

AU - Russo, I.

AU - Mattiello, L.

AU - Riganti, C.

AU - Anfossi, G.

AU - Trovati, M.

PY - 2006/5

Y1 - 2006/5

N2 - Aims/hypothesis: 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-1α 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-1α 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-1α 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-1α 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/interpretation: These results provide a biological mechanism for the impaired collateral vessel formation in obesity.

AB - Aims/hypothesis: 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-1α 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-1α 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-1α 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-1α 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/interpretation: These results provide a biological mechanism for the impaired collateral vessel formation in obesity.

KW - c-Jun N-terminal kinase

KW - Hypoxia-inducible factor

KW - Insulin

KW - Insulin resistance

KW - Mitogen-activated protein kinase

KW - Obesity

KW - Phosphatidylinositol-3 kinase

KW - Vascular endothelial growth factor

KW - Vascular smooth muscle

KW - Zucker rats

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U2 - 10.1007/s00125-006-0156-0

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JO - Diabetologia

JF - Diabetologia

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