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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 siRNA-mediated knockdown of APPL1 on insulin-stimulated ERK1/2 MAPK signaling cascades and ET-1 production in HUVECs. Cells were transfected with siRNA specific to APPL1 (siAPPL1) or scrambled control and then treated with insulin (50 nmol/L) for 10 min. Protein (40 μg) from total cell lysates was resolved by SDS-PAGE and probed for total ERK1/2 or phospho-ERK1/2 (A), total MEK1/2 and phospho-MEK1/2 (B), and total Raf-1 and phospho–Raf-1 at either Ser338 (C) or Ser259 (D). ET-1 concentration in the conditioned medium was measured 12 h after insulin treatment (E). T, total; p, phosphorylated. *P < 0.05, **P < 0.01 (n = 5–7).
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Figure 7: Effects of siRNA-mediated knockdown of APPL1 on insulin-stimulated ERK1/2 MAPK signaling cascades and ET-1 production in HUVECs. Cells were transfected with siRNA specific to APPL1 (siAPPL1) or scrambled control and then treated with insulin (50 nmol/L) for 10 min. Protein (40 μg) from total cell lysates was resolved by SDS-PAGE and probed for total ERK1/2 or phospho-ERK1/2 (A), total MEK1/2 and phospho-MEK1/2 (B), and total Raf-1 and phospho–Raf-1 at either Ser338 (C) or Ser259 (D). ET-1 concentration in the conditioned medium was measured 12 h after insulin treatment (E). T, total; p, phosphorylated. *P < 0.05, **P < 0.01 (n = 5–7).

Mentions: In contrast to the changes in the Akt/eNOS signaling, siRNA-mediated suppression of APPL1 expression in HUVECs resulted in a significant elevation in insulin-induced phosphorylation of ERK1/2 MAPK at Thr202/Tyr204 and an increase in phosphorylation of the mitogen-activated protein kinase kinase (MEK1/2) at Ser217/Ser221 (Fig. 7A and B). Further analysis of the upstream signaling events of MEK1/2 showed that knockdown of APPL1 expression caused a significant elevation in insulin-induced phosphorylation of Raf-1 at its activation site Ser338 (Fig. 7C) but an attenuated phosphorylation of Raf-1 at its inhibitory site Ser259 (Fig. 7D). On the other hand, suppression of APPL1 expression had no effect on the insulin-induced increase in activated, guanosine-5'-triphosphate (GTP)-bound Ras, an upstream activator of Raf-1 (data not shown). In parallel with the changes in ERK1/2 MAPK signaling, insulin-induced secretion of ET-1 in HUVECs was significantly increased by knockdown of APPL1 expression (Fig. 7E). Taken together, these findings suggest that APPL1 modulates insulin-induced ERK1/2 MAPK signaling and ET-1 production by altering the phosphorylation of Raf-1.


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 siRNA-mediated knockdown of APPL1 on insulin-stimulated ERK1/2 MAPK signaling cascades and ET-1 production in HUVECs. Cells were transfected with siRNA specific to APPL1 (siAPPL1) or scrambled control and then treated with insulin (50 nmol/L) for 10 min. Protein (40 μg) from total cell lysates was resolved by SDS-PAGE and probed for total ERK1/2 or phospho-ERK1/2 (A), total MEK1/2 and phospho-MEK1/2 (B), and total Raf-1 and phospho–Raf-1 at either Ser338 (C) or Ser259 (D). ET-1 concentration in the conditioned medium was measured 12 h after insulin treatment (E). 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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Figure 7: Effects of siRNA-mediated knockdown of APPL1 on insulin-stimulated ERK1/2 MAPK signaling cascades and ET-1 production in HUVECs. Cells were transfected with siRNA specific to APPL1 (siAPPL1) or scrambled control and then treated with insulin (50 nmol/L) for 10 min. Protein (40 μg) from total cell lysates was resolved by SDS-PAGE and probed for total ERK1/2 or phospho-ERK1/2 (A), total MEK1/2 and phospho-MEK1/2 (B), and total Raf-1 and phospho–Raf-1 at either Ser338 (C) or Ser259 (D). ET-1 concentration in the conditioned medium was measured 12 h after insulin treatment (E). T, total; p, phosphorylated. *P < 0.05, **P < 0.01 (n = 5–7).
Mentions: In contrast to the changes in the Akt/eNOS signaling, siRNA-mediated suppression of APPL1 expression in HUVECs resulted in a significant elevation in insulin-induced phosphorylation of ERK1/2 MAPK at Thr202/Tyr204 and an increase in phosphorylation of the mitogen-activated protein kinase kinase (MEK1/2) at Ser217/Ser221 (Fig. 7A and B). Further analysis of the upstream signaling events of MEK1/2 showed that knockdown of APPL1 expression caused a significant elevation in insulin-induced phosphorylation of Raf-1 at its activation site Ser338 (Fig. 7C) but an attenuated phosphorylation of Raf-1 at its inhibitory site Ser259 (Fig. 7D). On the other hand, suppression of APPL1 expression had no effect on the insulin-induced increase in activated, guanosine-5'-triphosphate (GTP)-bound Ras, an upstream activator of Raf-1 (data not shown). In parallel with the changes in ERK1/2 MAPK signaling, insulin-induced secretion of ET-1 in HUVECs was significantly increased by knockdown of APPL1 expression (Fig. 7E). Taken together, these findings suggest that APPL1 modulates insulin-induced ERK1/2 MAPK signaling and ET-1 production by altering the phosphorylation of Raf-1.

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