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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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Opposite effects of APPL1 on insulin-evoked Akt/eNOS signaling and ERK1/2 activation in mesenteric arteries. Arteries isolated from 16-week-old WT and APPL1-Tg mice on STD or HFD were treated with vehicle or insulin (20 nmol/L). A: Total tissue lysates were subjected to immunoblotting using the antibodies against total or phospho-Akt (Thr308), total or phospho-eNOS (Ser1177), and total or phospho-ERK1/2 (Thr202/Tyr204), as specified. The ratio of p-Akt/t-Akt (B), p-eNOS/t-eNOS (C), and p-ERK/t-ERK (D) were quantified by densitometry and are expressed as fold changes relative to the basal levels observed in preparations of WT mice on STD. T, total; p, phosphorylated. *P < 0.05, **P < 0.01 (n = 5–7).
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Figure 4: Opposite effects of APPL1 on insulin-evoked Akt/eNOS signaling and ERK1/2 activation in mesenteric arteries. Arteries isolated from 16-week-old WT and APPL1-Tg mice on STD or HFD were treated with vehicle or insulin (20 nmol/L). A: Total tissue lysates were subjected to immunoblotting using the antibodies against total or phospho-Akt (Thr308), total or phospho-eNOS (Ser1177), and total or phospho-ERK1/2 (Thr202/Tyr204), as specified. The ratio of p-Akt/t-Akt (B), p-eNOS/t-eNOS (C), and p-ERK/t-ERK (D) were quantified by densitometry and are expressed as fold changes relative to the basal levels observed in preparations of WT mice on STD. T, total; p, phosphorylated. *P < 0.05, **P < 0.01 (n = 5–7).

Mentions: In endothelial cells, insulin-evoked production of NO and ET-1 is mediated by the Akt/eNOS and the ERK1/2 MAPK signaling cascades, respectively (3,4). To investigate the impact of APPL1 on these two signaling pathways, mesenteric arteries isolated from 16-week-old WT and APPL1-Tg mice on either STD or HFD were exposed to vehicle or 20 nmol/L insulin for 10 min and collected for Western blotting (Fig. 4). The basal levels of eNOS phosphorylation at Ser1177 and Akt at Thr308 were similar between WT mice and APPL1-Tg mice on either STD or HFD. Insulin caused an increase in phosphorylation of both eNOS at Ser1177 and Akt at Thr308 in arteries of WT mice, and this stimulatory effect of the hormone was enhanced significantly in preparations of APPL1-Tg mice (Fig. 4B and C). The insulin-induced phosphorylation of both eNOS and Akt was significantly impaired in arteries of WT mice on HFD. The HFD-induced impairment in insulin-stimulated phosphorylation of Akt and eNOS was prevented by the transgenic expression of APPL1.


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)

Opposite effects of APPL1 on insulin-evoked Akt/eNOS signaling and ERK1/2 activation in mesenteric arteries. Arteries isolated from 16-week-old WT and APPL1-Tg mice on STD or HFD were treated with vehicle or insulin (20 nmol/L). A: Total tissue lysates were subjected to immunoblotting using the antibodies against total or phospho-Akt (Thr308), total or phospho-eNOS (Ser1177), and total or phospho-ERK1/2 (Thr202/Tyr204), as specified. The ratio of p-Akt/t-Akt (B), p-eNOS/t-eNOS (C), and p-ERK/t-ERK (D) were quantified by densitometry and are expressed as fold changes relative to the basal levels observed in preparations of WT mice on STD. T, total; p, phosphorylated. *P < 0.05, **P < 0.01 (n = 5–7).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198090&req=5

Figure 4: Opposite effects of APPL1 on insulin-evoked Akt/eNOS signaling and ERK1/2 activation in mesenteric arteries. Arteries isolated from 16-week-old WT and APPL1-Tg mice on STD or HFD were treated with vehicle or insulin (20 nmol/L). A: Total tissue lysates were subjected to immunoblotting using the antibodies against total or phospho-Akt (Thr308), total or phospho-eNOS (Ser1177), and total or phospho-ERK1/2 (Thr202/Tyr204), as specified. The ratio of p-Akt/t-Akt (B), p-eNOS/t-eNOS (C), and p-ERK/t-ERK (D) were quantified by densitometry and are expressed as fold changes relative to the basal levels observed in preparations of WT mice on STD. T, total; p, phosphorylated. *P < 0.05, **P < 0.01 (n = 5–7).
Mentions: In endothelial cells, insulin-evoked production of NO and ET-1 is mediated by the Akt/eNOS and the ERK1/2 MAPK signaling cascades, respectively (3,4). To investigate the impact of APPL1 on these two signaling pathways, mesenteric arteries isolated from 16-week-old WT and APPL1-Tg mice on either STD or HFD were exposed to vehicle or 20 nmol/L insulin for 10 min and collected for Western blotting (Fig. 4). The basal levels of eNOS phosphorylation at Ser1177 and Akt at Thr308 were similar between WT mice and APPL1-Tg mice on either STD or HFD. Insulin caused an increase in phosphorylation of both eNOS at Ser1177 and Akt at Thr308 in arteries of WT mice, and this stimulatory effect of the hormone was enhanced significantly in preparations of APPL1-Tg mice (Fig. 4B and C). The insulin-induced phosphorylation of both eNOS and Akt was significantly impaired in arteries of WT mice on HFD. The HFD-induced impairment in insulin-stimulated phosphorylation of Akt and eNOS was prevented by the transgenic expression of APPL1.

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