Akt Mediates the Effect of Insulin on Epithelial Sodium Channels by Inhibiting Nedd4-2

Discipline of Physiology, School of Medical Science, Faculty of Medicine, University of Sydney, Sydney, New South Wales 2006, Australia.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2007; 282(41):29866-73. DOI: 10.1074/jbc.M701923200
Source: PubMed


The epithelial sodium channel (ENaC) plays an important role in transepithelial Na+ absorption; hence its function is essential for maintaining Na+ and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this
study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum-
and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression
of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1K179M, decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not
only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin,
phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell
membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on
Nedd4-2 for Akt1 and Sgk1, Ser342 and Ser428, completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt
and Sgk are components of an insulin signaling pathway that increases Na+ absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.

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    • "We assume different ways of activation of ENaC by SGK1 and AKT, as several groups showed additional effects of both kinases (Lee et al. 2008; Deng et al. 2012). There are several regulatory mechanisms known for the insulin effect on ENaC, like translocation from intracellular pools to the plasma membrane (Blazer-Yost et al. 2003; Tiwari et al. 2007), preventing degradation by phosphorylation of Nedd4-2 (Lee et al. 2007) and activating the channel by direct phosphorylation (Shimkets et al. 1998; Zhang et al. 2005; Diakov et al. 2010) leading to an increased open probability (Tallini and Stoner 2002; Pavlov et al. 2013). Aside from that, basal control of ENaC function is strictly dependent on hormone levels, salt and water homeostasis and in vivo on stress and environmental conditions. "
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    ABSTRACT: Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19-day gestational age rat fetuses. Equivalent short-circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The ISC showed a fast dose-dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na+ channel) activity in experiments with permeabilized apical or basolateral membrane. 5-(N-Ethyl-N-isopropyl)amiloride inhibition of ISC was not affected, however, benzamil-sensitive ISC was increased in insulin-stimulated monolayers. The application of LY-294002 and Akti1/2 both completely blocked the stimulating effect of insulin on ISC. PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in ISC induced by insulin. The application of IGF-1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF-1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na+ transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells.
    Full-text · Article · Mar 2014
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    • "Since PKA is activated by cAMP, which is generated by vasopressin release and binding to its V2 receptor, it is likely that vasopressin, like aldosterone, can increase ENaC cell surface abundance by inhibiting Nedd4-2. In addition, the hormone insulin (Lee et al., 2007) and the kinases IKKB (Edinger et al., 2009) and with no lysine 1 (WNK1; Xu et al., 2005) were also shown to up-regulate ENaC via the Sgk1/Nedd4-2 pathway. In contrast, AMPK enhances the interactions between Nedd4-2 and ENaC, leading to increased ENaC endocytosis and channel downregulation under metabolic stress (Carattino et al., 2005; Bhalla et al., 2006; Almaca et al., 2009). "
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    ABSTRACT: Nedd4-2 is a ubiquitin ligase previously demonstrated to regulate endocytosis and lysosomal degradation of the epithelial Na(+) channel (ENaC) and other ion channels and transporters. Recent studies using Nedd4-2 knockout mice specifically in kidney or lung epithelia has revealed a critical role for this E3 ubiquitin ligase in regulating salt and fluid transport in these tissues/organs and in maintaining homeostasis of body blood pressure. Interestingly, the primary targets for Nedd4-2 may differ in these two organs: in the lung Nedd4-2 targets ENaC, and loss of Nedd4-2 leads to excessive ENaC function and to cystic fibrosis - like lung disease, whereas in the kidney, Nedd4-2 targets the Na(+)/Cl(-) cotransporter (NCC) in addition to targeting ENaC. In accord, loss of Nedd4-2 in the distal nephron leads to increased NCC abundance and function. The aldosterone-responsive kinase, Sgk1, appears to be involved in the regulation of NCC by Nedd4-2 in the kidney, similar to its regulation of ENaC. Collectively, these new findings underscore the physiological importance of Nedd4-2 in regulating epithelial salt and fluid transport and balance.
    Preview · Article · Jun 2012 · Frontiers in Physiology
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    • "In this study, we found that insulin-induced decrease in the levels of Nedd4-2 in coimmunoprecipitated α-ENaC, β- ENaC and γ-ENaC were blocked with LY294002 and Akt inhibitor treatment respectively These findings indicated the effect of insulin on inhibition of Nedd4-2 binding to ENaC via PI3K/Akt pathway. Recent study of Fisher rat thyroid cell proved the regulation of α-, β- and γ-ENaC heterologously expressed via PI3K/Akt pathway by suppression of Nedd4-2[60]. In addtion, co-administration of Akt inhibitor and SGK1 inhibitor, reported to inhibit SGK1[61], significantly inhibited insulin-induced increase in the expressions of α-, β- and γ-ENaC, as well as increased the insulin-induced decrease in the expression of Nedd4-2 compared with administration of Akt inhibitor alone in ATII cells. "
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    ABSTRACT: Stimulation of epithelial sodium channel (ENaC) increases Na(+) transport, a driving force of alveolar fluid clearance (AFC) to keep alveolar spaces free of edema fluid that is beneficial for acute lung injury (ALI). It is well recognized that regulation of ENaC by insulin via PI3K pathway, but the mechanism of this signaling pathway to regulate AFC and ENaC in ALI remains unclear. The aim of this study was to investigate the effect of insulin on AFC in ALI and clarify the pathway in which insulin regulates the expression of ENaC in vitro and in vivo. A model of ALI (LPS at a dose of 5.0 mg/kg) with non-hyperglycemia was established in Sprague-Dawley rats receiving continuous exogenous insulin by micro-osmotic pumps and wortmannin. The lungs were isolated for measurement of bronchoalveolar lavage fluid(BALF), total lung water content(TLW), and AFC after ALI for 8 hours. Alveolar epithelial type II cells were pre-incubated with LY294002, Akt inhibitor and SGK1 inhibitor 30 minutes before insulin treatment for 2 hours. The expressions of α-,β-, and γ-ENaC were detected by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin. Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC. In vitro, insulin increased the expressions of α-,β-, and γ-ENaC as well as the level of phosphorylated Akt but LY294002 and Akt inhibitor significantly prevented insulin-induced increase in the expression of ENaC and the level of phosphorylated Akt respectively. Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway. Our study demonstrated that insulin alleviated pulmonary edema and enhanced AFC by increasing the expression of ENaC that dependent upon PI3K/Akt pathway by inhibition of Nedd4-2.
    Full-text · Article · Mar 2012 · Respiratory research
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