Insulin activates epithelial sodium channel (ENaC) via phosphoinositide 3-kinase in mammalian taste receptor cells

Abstract

Diabetes is a profound disease that results in a severe lack of regulation of systemic salt and water balance. From our earlier work on the endocrine regulation of salt taste at the level of the epithelial sodium channel (ENaC), we have begun to investigate the ability of insulin to alter ENaC function with patch-clamp recording on isolated mouse taste receptor cells (TRCs). In fungiform and vallate TRCs that exhibit functional ENaC currents (e.g., amiloride-sensitive Na(+) influx), insulin (5-20 nM) caused a significant increase in Na(+) influx at -80 mV (EC(50) = 7.53 nM). The insulin-enhanced currents were inhibited by amiloride (30 μM). Similarly, in ratiometric Na(+) imaging using SBFI, insulin treatment (20 nM) enhanced Na(+) movement in TRCs, consistent with its action in electrophysiological assays. The ability of insulin to regulate ENaC function is dependent on the enzyme phosphoinositide 3-kinase since treatment with the inhibitor LY294002 (10 μM) abolished insulin-induced changes in ENaC. To test the role of insulin in the regulation of salt taste, we have characterized behavioral responses to NaCl using a mouse model of acute hyperinsulinemia. Insulin-treated mice show significant avoidance of NaCl at lower concentrations than the control group. Interestingly, these differences between groups were abolished when amiloride (100 μM) was added into NaCl solutions, suggesting that insulin was regulating ENaC. Our results are consistent with a role for insulin in maintaining functional expression of ENaC in mouse TRCs.

Fig. 1.

Effects of insulin on amiloride-sensitive cells. A: steady-state current in a mouse taste cell during whole cell patch-clamp recording. Shown is a continuous recording from a fungiform taste cell during the application of insulin (10 nM), insulin with amiloride (Amil; 30 μM), and insulin in Na⁺-free Tyrode's solution. Taste cells, like this one, showed a marked increase in sodium (Na⁺) inward current by the application of insulin. Solid line indicates timing of normal physiological saline. B: insulin dose-response curve. Insulin was applied by bath perfusion at 1, 3, 5, 7.5, 10, and 20 nM. Data points are means ± SE of the current enhancement as function of insulin concentration. Solid line is the statistically weighted best-fit logistic relation with EC₅₀ = 7.53 nM.

Fig. 2.

Functional sodium imaging confirms insulin enhancement of epithelial sodium channel (ENaC). Fungiform and circumvallate taste cells were preloaded with sodium-binding benzofuran isophthalate (SBFI) and Na⁺ mobilization was measured. A: SBFI ratio elicited by 140 mM NaCl delivered to fungiform taste cells in the presence or absence of insulin (20 nM) and subsequent application of amiloride (30 μM). B: circumvallate taste cells evoke greater responses to 140 mM Na⁺ in the presence of insulin. These insulin-mediated Na⁺ responses were generally inhibited by amiloride (30 μM). C: fungiform taste cells summary graph comparing the area under the curve of 140 mM NaCl responses before and after insulin (20 nM) application, as well as comparing the effects of amiloride (30 μM) treatment. D: summary graph comparing insulin effects in the posterior mouse tongue before and after amiloride (30 μM) application. Data are shown as means ± SE. ^a,cSignificant reduction in SBFI ratio; ^bsignificant increase in SBFI ratio compared with 140 mM NaCl (P < 0.01, ANOVA).

Fig. 3.

Insulin enhancement of ENaC-mediated Na⁺ transport occurs via phosphoinositide 3-kinase (PI3-K) signaling pathway. A: effects of PI3-K inhibitor LY294002 (10 μM) on insulin-induced Na⁺ responses in mouse fungiform taste cells using functional Na⁺ imaging with SBFI. B: functional Na⁺ imaging from circumvallate taste receptor cells showing changes in insulin enhancement of Na⁺ influx in the absence and presence of LY294002 (10 μM). C: summary graph of insulin effects in Na⁺ influx before and after LY303511 treatment. Data are shown as means ± SE. *Significant reduction in insulin-mediated Na⁺ influx by the PI3-K inhibitor (P < 0.01, Student's t-test).

Fig. 4.

Insulin-induced effects on amiloride-sensitive cells were abolished by both wortmannin and LY294002. A: insulin enhancement on Na⁺ influx was recorded with functional Na⁺ imaging in the absence and presence of wortmannin (1 μM) treatment for 1 h and before and after amiloride (30 μM). B: insulin-mediated responses in the absence and presence of pretreatment with wortmannin (50 nM) for 1 h and, subsequently, amiloride (30 μM). C: insulin-mediated responses in the absence and presence of acute treatment with LY294002 (10 μM) and, subsequently, amiloride (30 μM). All results obtained were compared with preceding 140 mM Na⁺ response. Values shown are means ± SE of between 8 and 10 cells per point.

Fig. 5.

The importance of serum- and glucocorticoid-regulated kinase (SGK) in insulin/ENaC mediated responses in taste cells. A: functional Na⁺ imaging with SBFI from SGK^+/+ wild-type mice in the presence of insulin (20 nM) and, subsequently, amiloride (30 μM). B: insulin effects on Na⁺ influx in SGK^−/− mice in the absence and presence of amiloride (30 μM) treatment. C: summary graph showing insulin-mediated effects in Na⁺ transport in SGK^+/+ wild-type animals. D: area under the curve graph from SGK^−/− mice showing a dramatic reduction in the magnitude of insulin-mediated Na⁺ responses. Data are shown as means ± SE. *Significant reduction in insulin-mediated Na⁺ influx by amiloride (30 μM; P < 0.01, Student's t-test).

Fig. 6.

RT-PCR reveals the presence of insulin receptor (IR; A), insulin receptor substrate 1 (IRS-1; B), and insulin receptor substrate 2 (IRS-2; C). Primers for IR, IRS-1, and IRS-2 amplify stained PCR products of expected sizes (IR 255 bp, IRS-1 491 bp, and IRS-2 296 bp). Positive controls (mouse kidney or liver RNA) are shown for IR, IRS-1, and IRS-2 with each set of primers. Negative control (−) lanes represent those in which cDNA was omitted from the PCR reaction.

Fig. 7.

Behavioral effects of insulin on salt preference appear to be via ENaC channels. A: NaCl/water lick ratio (means ± SE) measured in short-term taste assays using the Davis Rig in two groups of 22 mice. Insulin-treated mice significantly avoid NaCl solutions between 150 nM and 600 mM. *Significant difference in NaCl preference compared with control mice (P < 0.01, simple effects ANOVA). B: blood glucose levels (mg/dl) in each group. Values are means ± SE. C: NaCl/water lick ratio. Each point represents 12 mice for each group. Avoidance of NaCl solutions was reduced in both control and insulin-treated mice due to the blocking of ENaC channel by amiloride (100 μM), which was present in all solutions. Values are means ± SE. *Significant difference in NaCl preference compared with control mice (P < 0.01, simple effects ANOVA). D: graph of blood glucose levels (mg/dl) in each group. Values are mean ± SE. *Significant reduction in blood glucose levels by insulin treatment compared with control (P < 0.01, Student's t-test).