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APPL1 counteracts obesity-induced vascular insulin resistance and endothelial dysfunction by modulating the endothelial production of nitric oxide and endothelin-1 in mice.

Wang Y, Cheng KK, Lam KS, Wu D, Wang Y, Huang Y, Vanhoutte PM, Sweeney G, Li Y, Xu A - Diabetes (2011)

Bottom Line: However, the cellular mechanisms that control the dual vascular effects of insulin remain unclear.In endothelial cells, APPL1 potentiated insulin-stimulated Akt activation by competing with the Akt inhibitor Tribbles 3 (TRB3) and suppressed ERK1/2 signaling by altering the phosphorylation status of its upstream kinase Raf-1.APPL1 plays a key role in coordinating the vasodilator and vasoconstrictor effects of insulin by modulating Akt-dependent NO production and ERK1/2-mediated ET-1 secretion in the endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT

Objective: Insulin stimulates both nitric oxide (NO)-dependent vasodilation and endothelin-1 (ET-1)-dependent vasoconstriction. However, the cellular mechanisms that control the dual vascular effects of insulin remain unclear. This study aimed to investigate the roles of the multidomain adaptor protein APPL1 in modulating vascular actions of insulin in mice and in endothelial cells.

Research design and methods: Both APPL1 knockout mice and APPL1 transgenic mice were generated to evaluate APPL1's physiological roles in regulating vascular reactivity and insulin signaling in endothelial cells.

Results: Insulin potently induced NO-dependent relaxations in mesenteric arteries of 8-week-old mice, whereas this effect of insulin was progressively impaired with ageing or upon development of obesity induced by high-fat diet. Transgenic expression of APPL1 prevented age- and obesity-induced impairment in insulin-induced vasodilation and reversed obesity-induced augmentation in insulin-evoked ET-1-dependent vasoconstriction. By contrast, genetic disruption of APPL1 shifted the effects of insulin from vasodilation to vasoconstriction. At the molecular level, insulin-elicited activation of protein kinase B (Akt) and endothelial NO synthase and production of NO were enhanced in APPL1 transgenic mice but were abrogated in APPL1 knockout mice. Conversely, insulin-induced extracellular signal-related kinase (ERK)1/2 phosphorylation and ET-1 expression was augmented in APPL1 knockout mice but was diminished in APPL1 transgenic mice. In endothelial cells, APPL1 potentiated insulin-stimulated Akt activation by competing with the Akt inhibitor Tribbles 3 (TRB3) and suppressed ERK1/2 signaling by altering the phosphorylation status of its upstream kinase Raf-1.

Conclusions: APPL1 plays a key role in coordinating the vasodilator and vasoconstrictor effects of insulin by modulating Akt-dependent NO production and ERK1/2-mediated ET-1 secretion in the endothelium.

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Effects of transgenic expression of APPL1 on insulin-induced relaxation in mouse arteries. A: Schematic diagram of the transgenic construct. Shown is cDNA encoding FLAG-tagged full-length human APPL1 under the control of CMV-β-actin promoter. CMV, cytomegalovirus. B: Confirmation of the APPL1 transgenic expression by PCR using genomic DNA as a template. M, DNA marker. C: Western blot analysis to detect APPL1 expression using anti-APPL1 or anti-FLAG antibody. D: Relaxations to increasing concentrations of insulin in mesenteric arterial rings from 8-week-old APPL1-Tg and WT during U46619-induced contractions. Data are expressed as percentage of the contraction to U46619. EC, endothelium. E: Effects of the PI 3-K inhibitor LY294002 (5 μmol/L), the Akt inhibitor Akt-I-1 (5 μmol/L), the NOS inhibitor l-NAME (100 μmol/L), and removal of EC on insulin-induced relaxations of mesenteric arteries from both APPL1-Tg and WT littermates fed STD. Data are shown as area under the curve (AUC). *P < 0.05, **P < 0.01 (n = 6–8).
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Figure 1: Effects of transgenic expression of APPL1 on insulin-induced relaxation in mouse arteries. A: Schematic diagram of the transgenic construct. Shown is cDNA encoding FLAG-tagged full-length human APPL1 under the control of CMV-β-actin promoter. CMV, cytomegalovirus. B: Confirmation of the APPL1 transgenic expression by PCR using genomic DNA as a template. M, DNA marker. C: Western blot analysis to detect APPL1 expression using anti-APPL1 or anti-FLAG antibody. D: Relaxations to increasing concentrations of insulin in mesenteric arterial rings from 8-week-old APPL1-Tg and WT during U46619-induced contractions. Data are expressed as percentage of the contraction to U46619. EC, endothelium. E: Effects of the PI 3-K inhibitor LY294002 (5 μmol/L), the Akt inhibitor Akt-I-1 (5 μmol/L), the NOS inhibitor l-NAME (100 μmol/L), and removal of EC on insulin-induced relaxations of mesenteric arteries from both APPL1-Tg and WT littermates fed STD. Data are shown as area under the curve (AUC). *P < 0.05, **P < 0.01 (n = 6–8).

Mentions: In both mice and rats with obesity, APPL1 expression in mesenteric arteries is reduced (14,16). To investigate the physiological roles of APPL1 in modulating vascular reactivity, we generated transgenic mice with overexpression of FLAG-tagged human APPL1 (APPL1-Tg) under control of the β-actin promoter (Fig. 1A). Transgenic expression of human APPL1 in mesenteric arteries was confirmed by PCR (Fig. 1B) and Western blot analysis (Fig. 1C). In APPL1-Tg mice, APPL1 protein level in mesenteric arteries was ∼2.5-fold higher than in that in WT littermates. A similar expression pattern was also observed in carotid arteries and aorta (data not shown).


APPL1 counteracts obesity-induced vascular insulin resistance and endothelial dysfunction by modulating the endothelial production of nitric oxide and endothelin-1 in mice.

Wang Y, Cheng KK, Lam KS, Wu D, Wang Y, Huang Y, Vanhoutte PM, Sweeney G, Li Y, Xu A - Diabetes (2011)

Effects of transgenic expression of APPL1 on insulin-induced relaxation in mouse arteries. A: Schematic diagram of the transgenic construct. Shown is cDNA encoding FLAG-tagged full-length human APPL1 under the control of CMV-β-actin promoter. CMV, cytomegalovirus. B: Confirmation of the APPL1 transgenic expression by PCR using genomic DNA as a template. M, DNA marker. C: Western blot analysis to detect APPL1 expression using anti-APPL1 or anti-FLAG antibody. D: Relaxations to increasing concentrations of insulin in mesenteric arterial rings from 8-week-old APPL1-Tg and WT during U46619-induced contractions. Data are expressed as percentage of the contraction to U46619. EC, endothelium. E: Effects of the PI 3-K inhibitor LY294002 (5 μmol/L), the Akt inhibitor Akt-I-1 (5 μmol/L), the NOS inhibitor l-NAME (100 μmol/L), and removal of EC on insulin-induced relaxations of mesenteric arteries from both APPL1-Tg and WT littermates fed STD. Data are shown as area under the curve (AUC). *P < 0.05, **P < 0.01 (n = 6–8).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198090&req=5

Figure 1: Effects of transgenic expression of APPL1 on insulin-induced relaxation in mouse arteries. A: Schematic diagram of the transgenic construct. Shown is cDNA encoding FLAG-tagged full-length human APPL1 under the control of CMV-β-actin promoter. CMV, cytomegalovirus. B: Confirmation of the APPL1 transgenic expression by PCR using genomic DNA as a template. M, DNA marker. C: Western blot analysis to detect APPL1 expression using anti-APPL1 or anti-FLAG antibody. D: Relaxations to increasing concentrations of insulin in mesenteric arterial rings from 8-week-old APPL1-Tg and WT during U46619-induced contractions. Data are expressed as percentage of the contraction to U46619. EC, endothelium. E: Effects of the PI 3-K inhibitor LY294002 (5 μmol/L), the Akt inhibitor Akt-I-1 (5 μmol/L), the NOS inhibitor l-NAME (100 μmol/L), and removal of EC on insulin-induced relaxations of mesenteric arteries from both APPL1-Tg and WT littermates fed STD. Data are shown as area under the curve (AUC). *P < 0.05, **P < 0.01 (n = 6–8).
Mentions: In both mice and rats with obesity, APPL1 expression in mesenteric arteries is reduced (14,16). To investigate the physiological roles of APPL1 in modulating vascular reactivity, we generated transgenic mice with overexpression of FLAG-tagged human APPL1 (APPL1-Tg) under control of the β-actin promoter (Fig. 1A). Transgenic expression of human APPL1 in mesenteric arteries was confirmed by PCR (Fig. 1B) and Western blot analysis (Fig. 1C). In APPL1-Tg mice, APPL1 protein level in mesenteric arteries was ∼2.5-fold higher than in that in WT littermates. A similar expression pattern was also observed in carotid arteries and aorta (data not shown).

Bottom Line: However, the cellular mechanisms that control the dual vascular effects of insulin remain unclear.In endothelial cells, APPL1 potentiated insulin-stimulated Akt activation by competing with the Akt inhibitor Tribbles 3 (TRB3) and suppressed ERK1/2 signaling by altering the phosphorylation status of its upstream kinase Raf-1.APPL1 plays a key role in coordinating the vasodilator and vasoconstrictor effects of insulin by modulating Akt-dependent NO production and ERK1/2-mediated ET-1 secretion in the endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT

Objective: Insulin stimulates both nitric oxide (NO)-dependent vasodilation and endothelin-1 (ET-1)-dependent vasoconstriction. However, the cellular mechanisms that control the dual vascular effects of insulin remain unclear. This study aimed to investigate the roles of the multidomain adaptor protein APPL1 in modulating vascular actions of insulin in mice and in endothelial cells.

Research design and methods: Both APPL1 knockout mice and APPL1 transgenic mice were generated to evaluate APPL1's physiological roles in regulating vascular reactivity and insulin signaling in endothelial cells.

Results: Insulin potently induced NO-dependent relaxations in mesenteric arteries of 8-week-old mice, whereas this effect of insulin was progressively impaired with ageing or upon development of obesity induced by high-fat diet. Transgenic expression of APPL1 prevented age- and obesity-induced impairment in insulin-induced vasodilation and reversed obesity-induced augmentation in insulin-evoked ET-1-dependent vasoconstriction. By contrast, genetic disruption of APPL1 shifted the effects of insulin from vasodilation to vasoconstriction. At the molecular level, insulin-elicited activation of protein kinase B (Akt) and endothelial NO synthase and production of NO were enhanced in APPL1 transgenic mice but were abrogated in APPL1 knockout mice. Conversely, insulin-induced extracellular signal-related kinase (ERK)1/2 phosphorylation and ET-1 expression was augmented in APPL1 knockout mice but was diminished in APPL1 transgenic mice. In endothelial cells, APPL1 potentiated insulin-stimulated Akt activation by competing with the Akt inhibitor Tribbles 3 (TRB3) and suppressed ERK1/2 signaling by altering the phosphorylation status of its upstream kinase Raf-1.

Conclusions: APPL1 plays a key role in coordinating the vasodilator and vasoconstrictor effects of insulin by modulating Akt-dependent NO production and ERK1/2-mediated ET-1 secretion in the endothelium.

Show MeSH
Related in: MedlinePlus