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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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APPL1 KO mice display impaired relaxation and augmented ET-1–dependent contraction in response to insulin. Insulin-induced relaxations in mesenteric arteries of 8- and 16-week-old APPL1 KO and WT mice on STD. Data are expressed as percentage of the contraction to U46619 (A) or shown as area under the curve (AUC) (B). C: Effects of insulin in mesenteric arteries from 16-week-old APPL1 KO and WT mice fed HFD or STD in the presence of l-NAME (100 μmol/L). D: The mRNA levels of ET-1 in mesenteric arteries from 16-week-old APPL1 KO and WT mice fed STD or HFD were quantified using real-time PCR and normalized against GAPDH. *P < 0.05, **P < 0.01 (n = 5–7).
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Figure 5: APPL1 KO mice display impaired relaxation and augmented ET-1–dependent contraction in response to insulin. Insulin-induced relaxations in mesenteric arteries of 8- and 16-week-old APPL1 KO and WT mice on STD. Data are expressed as percentage of the contraction to U46619 (A) or shown as area under the curve (AUC) (B). C: Effects of insulin in mesenteric arteries from 16-week-old APPL1 KO and WT mice fed HFD or STD in the presence of l-NAME (100 μmol/L). D: The mRNA levels of ET-1 in mesenteric arteries from 16-week-old APPL1 KO and WT mice fed STD or HFD were quantified using real-time PCR and normalized against GAPDH. *P < 0.05, **P < 0.01 (n = 5–7).

Mentions: To investigate whether APPL1 is an indispensible regulator of insulin actions in the vasculature, we generated APPL1 KO mice by targeted disruption of the APPL1 gene at exon 17–18 (Supplementary Fig. 2A). PCR analysis was used to genotype mutant mice (Supplementary Fig. 2B). Western blotting confirmed the absence of APPL1 protein expression in APPL1 KO mice (Supplementary Fig. 2C). The relaxation of mesenteric arteries of APPL1 KO mice to insulin were impaired significantly compared with age-matched WT preparations, even when they were fed STD (Fig. 5A and B). The maximal relaxation to 100 nmol/L insulin in APPL1 KO and WT arteries was, respectively, 36.3 ± 5.8% and 71.6 ± 3.43% (P < 0.01) at the age of 8 weeks and 28.7 ± 7.7% and 56.8 ± 3.13% (P < 0.05) at the age of 16 weeks. In the presence of l-NAME, 100 nmol/L insulin caused a modest contraction in arteries of APPL1 KO mice even when they were fed STD (Fig. 5C). When fed HFD, the insulin-elicited increase in tension was significantly stronger in preparations of APPL1 KO than that in those of WT littermates (Fig. 5C). With both STD and HFD, the magnitude of insulin-induced increase in ET-1 expression in APPL1 KO was significantly higher than that in WT arteries (Fig. 5D). APPL1 KO mice exhibited significantly elevated systolic blood pressure when the mice aged beyond 16 weeks (data not shown). In 20-week-old APPL1 KO mice fed with HFD, systolic blood pressure was 10.5% higher than age-matched WT littermates. Western blotting analysis demonstrated that insulin-stimulated phosphorylation of eNOS at Ser1177 and Akt at Thr308 in mesenteric arteries of APPL1 KO mice was significantly blunted compared with WT controls (Supplementary Fig. 3). By contrast, insulin-stimulated ERK1/2 phosphorylation was elevated in arteries of APPL1 KO mice.


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)

APPL1 KO mice display impaired relaxation and augmented ET-1–dependent contraction in response to insulin. Insulin-induced relaxations in mesenteric arteries of 8- and 16-week-old APPL1 KO and WT mice on STD. Data are expressed as percentage of the contraction to U46619 (A) or shown as area under the curve (AUC) (B). C: Effects of insulin in mesenteric arteries from 16-week-old APPL1 KO and WT mice fed HFD or STD in the presence of l-NAME (100 μmol/L). D: The mRNA levels of ET-1 in mesenteric arteries from 16-week-old APPL1 KO and WT mice fed STD or HFD were quantified using real-time PCR and normalized against GAPDH. *P < 0.05, **P < 0.01 (n = 5–7).
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Figure 5: APPL1 KO mice display impaired relaxation and augmented ET-1–dependent contraction in response to insulin. Insulin-induced relaxations in mesenteric arteries of 8- and 16-week-old APPL1 KO and WT mice on STD. Data are expressed as percentage of the contraction to U46619 (A) or shown as area under the curve (AUC) (B). C: Effects of insulin in mesenteric arteries from 16-week-old APPL1 KO and WT mice fed HFD or STD in the presence of l-NAME (100 μmol/L). D: The mRNA levels of ET-1 in mesenteric arteries from 16-week-old APPL1 KO and WT mice fed STD or HFD were quantified using real-time PCR and normalized against GAPDH. *P < 0.05, **P < 0.01 (n = 5–7).
Mentions: To investigate whether APPL1 is an indispensible regulator of insulin actions in the vasculature, we generated APPL1 KO mice by targeted disruption of the APPL1 gene at exon 17–18 (Supplementary Fig. 2A). PCR analysis was used to genotype mutant mice (Supplementary Fig. 2B). Western blotting confirmed the absence of APPL1 protein expression in APPL1 KO mice (Supplementary Fig. 2C). The relaxation of mesenteric arteries of APPL1 KO mice to insulin were impaired significantly compared with age-matched WT preparations, even when they were fed STD (Fig. 5A and B). The maximal relaxation to 100 nmol/L insulin in APPL1 KO and WT arteries was, respectively, 36.3 ± 5.8% and 71.6 ± 3.43% (P < 0.01) at the age of 8 weeks and 28.7 ± 7.7% and 56.8 ± 3.13% (P < 0.05) at the age of 16 weeks. In the presence of l-NAME, 100 nmol/L insulin caused a modest contraction in arteries of APPL1 KO mice even when they were fed STD (Fig. 5C). When fed HFD, the insulin-elicited increase in tension was significantly stronger in preparations of APPL1 KO than that in those of WT littermates (Fig. 5C). With both STD and HFD, the magnitude of insulin-induced increase in ET-1 expression in APPL1 KO was significantly higher than that in WT arteries (Fig. 5D). APPL1 KO mice exhibited significantly elevated systolic blood pressure when the mice aged beyond 16 weeks (data not shown). In 20-week-old APPL1 KO mice fed with HFD, systolic blood pressure was 10.5% higher than age-matched WT littermates. Western blotting analysis demonstrated that insulin-stimulated phosphorylation of eNOS at Ser1177 and Akt at Thr308 in mesenteric arteries of APPL1 KO mice was significantly blunted compared with WT controls (Supplementary Fig. 3). By contrast, insulin-stimulated ERK1/2 phosphorylation was elevated in arteries of APPL1 KO mice.

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