Insulin increases the turnover rate of Na+-K+-ATPase in human fibroblasts

Nicola Longo, Fernando Scaglia, and Yuhuan Wang

Division of Medical Genetics, Department of Pediatrics, Emory University, Atlanta, Georgia 30322


    ABSTRACT
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MATERIALS AND METHODS
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Insulin stimulates K+ transport by the Na+-K+-ATPase in human fibroblasts. In other cell systems, this action represents an automatic response to increased intracellular [Na+] or results from translocation of transporters from an intracellular site to the plasma membrane. Here we evaluate whether these mechanisms are operative in human fibroblasts. Human fibroblasts expressed the alpha 1 but not the alpha 2 and alpha 3 isoforms of Na+-K+-ATPase. Insulin increased the influx of Rb+, used to trace K+ entry, but did not modify the total intracellular content of K+, Rb+, and Na+ over a 3-h incubation period. Ouabain increased intracellular Na+ more rapidly in cells incubated with insulin, but this increase followed insulin stimulation of Rb+ transport. Bumetanide did not prevent the increased Na+ influx or stimulation of Na+-K+-ATPase. Stimulation of the Na+-K+- ATPase by insulin did not produce any measurable change in membrane potential. Insulin did not affect the affinity of the pump toward internal Na+ or the number of membrane-bound Na+-K+-ATPases, as assessed by ouabain binding. By contrast, insulin slightly increased the affinity of Na+-K+-ATPase toward ouabain. Phorbol esters did not mimic insulin action on Na+-K+-ATPase and inhibited, rather than stimulated, Rb+ transport. These results indicate that insulin increases the turnover rate of Na+-K+-ATPases of human fibroblasts without affecting their number on the plasma membrane or modifying their dependence on intracellular [Na+].

membrane transport; phorbol esters; membrane potential; rubidium transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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INSULIN INITIATES ITS ACTION by interacting with specific membrane receptors located on the plasma membrane of target cells. The insulin-receptor complex activates a number of cellular functions, among which is transmembrane transport of ions and nutrients. In human fibroblasts, insulin stimulates the transport of potassium, glucose, and amino acids (11, 13, 14, 17). The stimulation of potassium transport is due to activation of both the Na+-K+-Cl- cotransporter and the Na+-K+-ATPase; it occurs very rapidly and reaches a maximum after only 10 min (13). By contrast, insulin stimulation of glucose and amino acid transport requires significantly longer times and reaches a maximum after 30 min and several hours, respectively (14, 17). The mechanism by which insulin stimulates the Na+-K+-ATPase in human fibroblasts is not known.

The Na+-K+-ATPase is composed of an alpha - (112 kDa) and a beta -subunit (35 kDa). The alpha -subunit contains the binding sites for Na+, K+, ATP, and ouabain and is commonly referred to as the catalytic subunit. There are four isoforms of Na+-K+-ATPase alpha -subunits (alpha 1, alpha 2, alpha 3, and alpha 4) (29). The beta -subunit is a glycoprotein whose function, although essential to pump activity, remains to be determined (29). There are at least three isoforms of Na+-K+-ATPase beta -subunit (beta 1, beta 2, and beta 3). All these different isoforms have different tissue-specific expression (29), with alpha 4 being expressed only in the testis (32, 33).

The mechanism of insulin stimulation of Na+-K+-ATPase activity varies among different cells and tissues. In rat hepatocytes (9) and BC3H-1 myocytes (26), insulin stimulates pump activity by increasing the availability of Na+ in the cytoplasm. Increased Na+ availability is due to increased Na+ influx through the amiloride-sensitive Na+/H+ exchanger (9, 26). In 3T3-L1 fibroblasts, increased Na+ entry through the bumetanide-sensitive Na+-K+-Cl- cotransporter has been proposed to play a major role in insulin stimulation of Na+-K+-ATPase, since bumetanide prevents the stimulation (30). In 3T3-F442A fibroblasts and adipocytes, insulin stimulates pump activity again by increasing Na+ entry, but this time through a Na+ channel, which is not inhibited by amiloride (4). This channel may correspond to the µ-conotoxin-sensitive Na+ channel reported in skeletal muscle (20). It is not known whether increased Na+ entry raises intracellular [Na+], since total intracellular [Na+] has not been measured. In addition, it is unclear whether the eventual increase in intracellular [Na+] precedes the insulin effect on Na+-K+-ATPase.

In primary adipocytes, insulin does not increase Na+ entry (21) and directly stimulates the alpha 2 isoform of the pump by increasing its affinity toward intracellular Na+ (18). In these cells, insulin does not increase the number of functional Na+-K+-ATPases on the plasma membrane (25). By contrast, in the skeletal muscle, another tissue that expresses the alpha 2 isoform of Na+-K+-ATPase, insulin increases the number of Na+-K+-ATPases in the plasma membrane (10, 19). Immunological studies have indicated that insulin specifically recruits preformed alpha 2 and beta 1 isoforms, but not alpha 1 isoforms, from an intracellular pool (10), in analogy with the mechanism of insulin action on the insulin-responsive glucose transporter (GLUT4) (10). It is not clear why the same isoform of Na+-K+-ATPases behaves differently in different tissues.

In this paper, we report that insulin increases the activity of the Na+-K+-ATPase of human fibroblasts without increasing intracellular [Na+] or the affinity of the pump toward intracellular [Na+]. Insulin effect occurs without an increase in the number of membrane-associated ouabain-binding sites, indicating that insulin increases the turnover rate of existing transporters.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Materials. 3-O-[U-14C]methyl-D-glucose (55 mCi/mmol) was from NEN. L-[2,3,4,5-3H]arginine monohydrochloride (57 Ci/mmol) and [21,22-3H]ouabain (32 Ci/mmol) were from Amersham. Insulin (bovine sodium, 25 U/mg) was from Calbiochem. Chemical reagents were American Chemical Society grade and were obtained from Sigma or Fisher.

Experimental techniques. Human fibroblasts were cultured in Dulbecco-Vogt medium containing 15% fetal bovine serum. For experiments, cells were seeded in 24-well plates and grown to confluence, and the medium was renewed every 3 days as well as 48 h before each experiment. On the day of the experiment, cells were washed twice and incubated for 2 h at 37°C in Tris (26 mM, pH 7.4)-buffered Earle's balanced salt solution (EBSS) containing 116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM NaH2PO4, 0.8 mM MgSO4, and 5.5 mM D-glucose, supplemented with 1% (wt/vol) bovine serum albumin (RIA grade; Sigma). Insulin was then added to the cells, and nonradioactive Rb+ uptake was then measured for the time indicated in the presence or absence of ouabain or bumetanide. In the uptake solution, 5.4 mM KCl was replaced by 5.4 mM RbCl (Rb-EBSS) (16). Cell monolayers were rapidly washed four times with ice-cold 0.1 M MgCl2 (total washing time <10 s) (16). Ethanol (0.1 ml) was then added to each well and allowed to dry. Two milliliters of 5 mM CsCl in water were added to each well, and intracellular Na+, Rb+, and K+ contents were determined in each well by emission flame photometry (Perkin Elmer 460 Atomic Absorption Spectrophotometer). Cell monolayers were then dried, solubilized with 200 µl of 0.1% sodium deoxycholate in 1 M NaOH, and assayed for protein by using a modified Lowry procedure (31).

The membrane potential of human fibroblasts was estimated from the distribution ratio of L-arginine (5). Fibroblasts were incubated for 1 h in EBSS containing L-[3H]arginine (20 µM, 2 µCi/ml) in the absence or presence of insulin, ouabain, or valinomycin. Cells were then washed three times with ice-cold 0.1 M MgCl2, and intracellular arginine was extracted in 0.5 ml of ethanol. The ethanol extract was added to 3 ml of scintillation fluid and counted in a scintillation counter. Intracellular arginine concentration was determined by dividing for intracellular water. Membrane potential was calculated by applying the Nernst equation to the distribution ratio of L-arginine (5).

Ouabain binding was measured for 30 min at 37°C. Cells were incubated for 10 min in EBSS without or with insulin, and then they were washed twice with EBSS in which KCl was replaced by choline chloride and incubated with or without insulin in the same K+-free solution for 30 min in the presence of increasing concentrations of ouabain (4-500 nM). Nonspecific binding was measured in the presence of 1 mM cold ouabain and accounted for <2% of total binding at 4 nM ouabain. After binding, cells were washed three times with ice-cold 0.1 M MgCl2, and membrane-bound radioactivity was determined by extraction in ethanol, as for arginine accumulation. Bound ouabain was then normalized to protein content. Preliminary experiments in human fibroblasts indicated that extracellular K+ inhibited ouabain binding with half-maximal inhibition observed at 1.8 ± 0.4 mM. In the absence of extracellular K+, equilibrium ouabain binding was reached at 30 min and remained constant up to 120 min (not shown).

Calculations. Intracellular water was evaluated in parallel experiments from the equilibrium distribution of 3-O- methyl-D-glucose, which is reached within 10 min in cultured human fibroblasts (15). The mean cell water content was 7.0 ± 0.7 µl/mg of cell proteins in human fibroblasts and was not affected by insulin treatment.

Intracellular ion content was calculated from the total amount of nonradioactive Na+, K+, and Rb+ (as determined by flame photometry) and was then expressed as micromoles per milliliter of cell water by dividing for the corresponding intracellular water space. Ouabain-sensitive Rb+ uptake was determined by subtracting the uptake in the presence of the inhibitor from total Rb+ uptake (16). The standard error of the difference between two samples was determined as the square root of the sum of the two sample variances (16).

Statistical comparisons were performed by using analysis of variance. The analysis of kinetic curves was performed by nonlinear regression analysis with the use of SigmaPlot. The equation used for the uptake of Rb+ at different intracellular [Na+] was
v=<FR><NU>V<SUB>max</SUB><IT>·</IT>[Na<SUP>+</SUP>]<SUP><IT>3</IT></SUP></NU><DE>(<IT>K<SUB>0.5</SUB></IT>)<SUP><IT>3</IT></SUP><IT>+</IT>[Na<SUP>+</SUP>]<SUP><IT>3</IT></SUP></DE></FR> (1)
This equation assumes three highly cooperative binding sites (v is the measured activity of Na+-K+-ATPase, Vmax is the maximal velocity at saturating concentrations of intracellular Na+, and K0.5 is a measure of Na+ affinity). These equations assume that, at each cycle, the pump moves out three Na+ ions (18). The stoichiometry of the Na+-K+- ATPase of human fibroblasts is compatible with such a model (16). This analysis intended not to evaluate the mechanism of Na+ interaction with the Na+-K+-ATPase but only to obtain quantitative parameters for the dependence of Rb+ transport on intracellular [Na+] in control and insulin-stimulated cells (18). Kinetics of ouabain binding were fitted to the equation
Bound<IT>=</IT><FR><NU>B<SUB>max</SUB><IT>·</IT>[Ouabain]</NU><DE><IT>K</IT><SUB>d</SUB><IT>+</IT>[Ouabain]</DE></FR> (2)
where Bmax is the maximal binding and Kd is the concentration of ouabain at which half-maximal binding is observed.

Nonlinear parameters are expressed in the text as means ± 95% confidence intervals.

RNA analysis. RNA was extracted from confluent cells or from tissues by using guanidinium thiocyanate (6). Total RNA (5-20 µg) was separated by formaldehyde-agarose gel electrophoresis, transferred to nylon (ZetaProbe; Bio-Rad), and hybridized in high-stringency conditions to cDNA encoding the rat alpha 1 (full length), alpha 2 (1.8-kb fragment), and alpha 3 (278-bp PstI-SmaI fragment corresponding to the 5' of the cDNA) isoforms of Na+-K+-ATPase labeled with [32P]dCTP by primer extension. Blots were then washed at 65°C in 0.5× standard sodium citrate and autoradiographed for 16-24 h at -70°C.


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Northern blot analysis. In rats, there are four isoforms of the alpha -subunit of Na+-K+-ATPase, with alpha 4 being expressed only in testis and essential for sperm motility (32, 33). To define which of the other three isoforms of Na+-K+-ATPase were present in human fibroblasts, we hybridized RNA to cDNAs coding for the alpha 1, alpha 2, and alpha 3 isoforms of Na+-K+-ATPase. Human fibroblasts expressed the alpha 1 isoform and had a band of 3.8 kb (Fig. 1). Human fibroblasts did not have mRNA for the alpha 2 isoform of Na+-K+-ATPase, while RNA from bovine brain (used as positive control) gave hybridizing bands of 5.4 and 3.5 kb. Bovine brain, but not human fibroblasts, expressed a 3.7-kb mRNA corresponding to the alpha 3 isoform of Na+-K+-ATPase.


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Fig. 1.   Expression of the alpha 1 isoform of Na+-K+-ATPase in human fibroblasts. Total RNA was isolated from confluent fibroblast cultures and bovine brain, separated by formaldehyde-agarose gel electrophoresis, blotted to nylon, and hybridized in high-stringency conditions to rat cDNA for the alpha 1, alpha 2, and alpha 3 isoforms of Na+-K+-ATPase. The agarose gel is shown below each blot for comparison. We used 5 µg of RNA from bovine brain and 20 µg of RNA from human fibroblasts.

Effect of insulin on intracellular ion concentrations. Figure 2 shows the changes in intracellular [Na+], [K+], and [Rb+] in human fibroblasts incubated in a buffered solution containing Rb+ instead of K+. As expected (16), Rb+ replaced K+ in the intracellular space (Fig. 2A). The sum of [Rb+] and [K+] remained constant over time, as did intracellular [Na+]. In the presence of insulin (500 nM; Fig. 2B), the exchange of Rb+ for K+ was faster. However, the sum of [Rb+] and [K+] remained constant and was not significantly different from that measured in the absence of insulin.


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Fig. 2.   Effect of insulin on intracellular cation concentration. Cells were incubated for up to 3 h at 37°C in the absence (A, C, E, and G) or presence (B, D, F, and H) of insulin (500 nM) in Rb+-containing medium. Insulin, when present, was added 10 min before and during the uptake assay. Ouabain (0.4 mM; C and D), bumetanide (0.1 mM; E and F), and both bumetanide and ouabain (G and H) were also present during the incubation. At the end of the incubation period, intracellular Na+, K+, and Rb+ contents were then determined by flame photometry and corrected for intracellular water, determined in a parallel tray from the equilibrium distribution of 3-O-[U14C]methyl-D-glucose. Data are means ± SD of triplicates. The experiment was repeated three times with similar results.

When ouabain (0.4 mM) was added to the cells (Fig. 2, C and D) the sum of [Rb+] and [K+] declined over time, while intracellular [Na+] increased. Insulin (Fig. 2D) increased the rate of decrease in intracellular [K+] and the rate of increase in intracellular [Na+].

Bumetanide (0.1 mM) slightly decreased the rate at which Rb+ replaced intracellular K+ and had no effect on intracellular [Na+] (Fig. 2E). Insulin (Fig. 2F) added with bumetanide stimulated the rate of exchange between Rb+ and K+ above that measured with (Fig. 2E) or without (Fig. 2A) bumetanide. However, Rb+/K+ exchange was slowed by bumetanide compared with that in cells treated with insulin alone (Fig. 2B).

When ouabain and bumetanide were added together (Fig. 2G), the sum of intracellular [Rb+] and [K+] fell more rapidly than when ouabain was added alone (Fig. 2C). This was due to markedly decreased Rb+ entry, since the decrease in intracellular [K+] was actually faster in cells treated with ouabain alone. Intracellular [Na+] increased in cells treated with bumetanide and ouabain similarly to increases in cells treated with ouabain alone. The addition of insulin (Fig. 2H) increased the rate of intracellular [Na+] accumulation and the rate of decrease of the sum of intracellular [K+] and [Rb+].

Ouabain-sensitive Rb+ and ouabain-induced Na+ accumulations by fibroblasts incubated in the absence or presence of insulin were estimated from the differences between cells incubated in the absence and presence of ouabain. The results are reported in Fig. 3. As expected, insulin increased ouabain-sensitive Rb+ accumulation by cultured fibroblasts, and its effect was significant at the first time point measured (10 min) (Fig. 3A). Bumetanide did not decrease this action of insulin. Insulin increased ouabain-induced Na+ uptake by human fibroblasts in the absence or presence of bumetanide (Fig. 3B). However, the increase in ouabain-induced Na+ uptake became significant only after 30 min and followed, rather than preceded, the increase in Rb+ uptake.


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Fig. 3.   Effect of insulin on ouabain-dependent Rb+ (A) and Na+ (B) accumulation. Ouabain-sensitive Rb+ and Na+ uptake were calculated by differences between data in Fig. 2, A and C (basal); B and D (500 nM insulin); E and G (0.1 mM bumetanide); and F and H (bumetanide + insulin). Data are means ± SE of triplicates.

Effect of insulin on the membrane potential of human fibroblasts. Insulin modifies the membrane potential of a number of cell types (23). It is not known whether this applies to fibroblasts. Figure 4 shows the effect of insulin (500 nM) on the membrane potential of human fibroblasts. Valinomycin (20 µM), which increases K+ permeability and the membrane potential (5), and ouabain (0.4 mM), which decreases intracellular K+ concentration and the membrane potential (16), were used as internal controls. A 1-h incubation in the presence of insulin did not affect the membrane potential. Shorter and longer incubation times (30-120 min) with insulin also failed to modify the membrane potential. These data are consistent with the lack of changes in intracellular ion content shown in Fig. 2.


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Fig. 4.   Effect of insulin on the membrane potential of human fibroblasts. Cells were incubated for 1 h with 20 µM arginine in the absence (basal) or presence of insulin (500 nM), ouabain (0.4 mM), or valinomycin (20 µM). Insulin, when present, was added 10 min before and during the incubation with arginine. The membrane potential was estimated by applying the Nernst equation to the distribution ratio of arginine. Data are means ± SD of triplicates. The experiment was repeated twice with similar results.

Mechanism for insulin stimulation of Na+-K+- ATPase in human fibroblasts. In primary adipocytes, insulin stimulates pump activity by increasing the affinity of Na+-K+-ATPase toward internal Na+ (18). Internal [Na+] was varied by incubating human fibroblasts during the 5-min uptake assay with increasing concentrations of Na+ (1-120 mM) in the presence of monensin, a Na+ ionophore (5 µg/ml). Na+ was replaced by choline in the incubation medium such that the sum of [Na+] and [choline+] remained at 120 mM. Intracellular Na+ was determined by flame photometry and corrected for the intracellular water content, determined in parallel trays from the equilibrium distribution of 3-O-methyl-D-glucose (15). Rb+ uptake was determined in both the absence and presence of 1 mM ouabain to calculate the portion of Rb+ uptake due to Na+-K+-ATPase. Incubation of human fibroblasts with monensin and different extracellular [Na+] varied intracellular [Na+] from 10 to 50 mM (Fig. 5). Pump activity in cells incubated in the absence of insulin was highly dependent on intracellular [Na+] with a K0.5 of 23.1 ± 2.8 mM, assuming a cooperative model. Addition of insulin did not affect the K0.5, which remained at 19.9 ± 2.0 mM. The difference between control and insulin-stimulated cells was not significant (P > 0.05) when 95% confidence intervals were used. Rb+ uptake was always higher in insulin-stimulated cells at all intracellular [Na+] created by the experimental procedure inside the cell. The estimated maximal transport activity by Na+-K+-ATPase at 5.4 mM Rb+ was 3.4 ± 0.4 and 4.9 ± 0.4 µmol · ml cell water-1 · min-1 (P < 0.01) in the absence and presence of insulin, respectively. This finding indicates that insulin-stimulated cells have an increased number or activity of Na+-K+-ATPases.


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Fig. 5.   Dependence of the Na+-K+ pump on intracellular [Na+]: effect of insulin. Cells were incubated during the 5-min influx assay in a medium containing 1, 3, 5, 9, 16, 30, 60, and 117 mM Na+ and 5 µg/ml (7.5 µM) monensin. During this period, Rb+ influx was measured in the absence (basal) and presence of ouabain (1 mM) and insulin (500 nM). Insulin, when present, was added 10 min before and during the uptake assay. With monensin, the presence of ouabain only minimally affected intracellular [Na+] ([Na+]in). Concentration values are means ± SD of 6 determinations (3 without and 3 with ouabain). Rb+ and Na+ contents were both measured in the same sample. Insulin did not affect the affinity of the pump toward internal Na+ (K0.5): basal, 23.1 ± 1.4 mM; insulin, 19.9 ± 1.0 mM (P > 0.05). Ouabain-sensitive Rb+ influx is the difference between total Rb+ influx and Rb+ influx in the presence of the cardiac glycoside, and influx values are means ± SE of triplicates. In a parallel tray, water content was measured in each condition. Lines represent the best fit of data to Eq. 1 (see MATERIALS AND METHODS).

To determine whether insulin recruited or unmasked Na+-K+-ATPases from an intracellular site, we measured ouabain binding to fibroblasts treated or not treated with insulin (Fig. 6). Equilibrium ouabain binding was similar in cells treated or not treated with insulin. The maximal binding capacity was identical (4.3 ± 0.2 pmol/mg cell protein), but insulin decreased the Kd for binding from 52.3 ± 6.8 to 35.9 ± 4.8 nM (P < 0.05). This result indicates that insulin stimulates the Na+-K+-ATPase of human fibroblasts without increasing the number of membrane-associated pumps.


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Fig. 6.   Effect of insulin on equilibrium ouabain binding by human fibroblasts. A: cells were incubated for 30 min in the presence of increasing concentrations of ouabain (4-500 nM). Nonspecific binding, measured in the presence of 1 mM cold ouabain, was <2% of total binding at 4 nM ouabain and was subtracted from all values before data were plotted. Insulin, when present, was added 10 min before and during the binding assay. Data are averages of triplicates. The experiment was repeated twice with similar results. Lines represent the best fit of data to a Michaelis-Menten equation. B: ouabain binding in a Scatchard graphical representation. Insulin stimulation of Rb+ uptake by the Na+-K+-ATPase in parallel cells was 63% above basal level. Basal: maximal binding (Bmax) = 4.3 ± 0.2 pmol/mg cell protein, and the concentration of ouabain at which half-maximal binding occurs (Kd) = 52.3 ± 6.8 nM. Insulin: Bmax = 4.3 ± 0.2 pmol/mg cell protein, Kd = 35.9 ± 4.8 nM.

Effect of kinase activators and inhibitors on insulin stimulation of Rb+ influx. Insulin interacts with specific receptors on the plasma membrane of target cells and stimulates their phosphorylation on tyrosine residues and kinase activity toward endogenous substrates. Previous studies (13) indicated that genistein, a tyrosine-kinase inhibitor, and staurosporine, a serine/threonine kinase inhibitor, significantly reduced insulin stimulation of Na+-K+-ATPase. These results indicate that the kinase activity of the insulin receptor is required to stimulate ion transport and that a serine/threonine kinase is also involved. Phorbol esters mimic several actions of insulin, including stimulation of glucose transport in human fibroblasts (17). Incubation of human fibroblasts with phorbol 12,13-dibutyrate (PDBU) at a concentration capable of fully stimulating glucose transport (17) inhibited, rather than stimulated, ouabain-sensitive Rb+ influx (Fig. 7). Insulin reversed the inhibition caused by phorbol esters.


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Fig. 7.   Effect of phorbol 12,13-dibutyrate (PDBU) and wortmannin on insulin stimulation of Rb+ influx. A: fibroblasts were incubated for 5 min in the presence of insulin, PDBU, or PDBU + insulin, after which Rb+ influx was measured for 5 min with or without ouabain (0.4 mM). B: wortmannin, when present, was added 10 min before insulin. Ouabain-sensitive Rb+ influx was calculated by differences (indicated by values above bars) between total influx and influx in the presence of the inhibitor. Data are means ± SE of 3 observations.

Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocks insulin stimulation of Rb+ influx in 3T3-L1 fibroblasts (30). In human fibroblasts, 0.1 µM wortmannin, a concentration inhibiting insulin stimulation of glucose transport (22), did not prevent insulin stimulation of Rb+ influx without affecting significantly basal pump activity (Fig. 7B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Insulin interacts with specific receptors on the plasma membrane of target cells and stimulates a number of cellular functions, including potassium (Rb+) transport. In human fibroblasts, insulin stimulates Rb+ transport by both the Na+-K+-Cl- cotransporter and the Na+-K+-ATPase (13). These effects of insulin are very rapid and precede stimulation of glucose and amino acid transport (14, 17). The specific isoform of Na+-K+-ATPase expressed in human fibroblasts and how it is stimulated by insulin is not known. Northern blot analysis indicated that human fibroblasts express only the alpha 1 isoform of Na+-K+-ATPase, while mRNA for the alpha 2 and alpha 3 isoforms was not present (Fig. 1). The alpha 2 isoform of Na+-K+-ATPase was found to be translocated to the plasma membrane in response to insulin in the muscle (10) or to increase its affinity toward intracellular Na+ in adipocytes (18). By contrast, the alpha 1 isoform of Na+-K+-ATPase is usually expressed in other fibroblast-like cells, and in cells other than human fibroblasts, its activity is stimulated by increased intracellular [Na+] caused by hormonal exposure (9, 26).

Previous studies (13) indicate that activation of Na+-K+-ATPase by insulin is dose dependent and increases the Vmax of the pump without significantly affecting the Michaelis-Menten constant (Km) toward K+. This could be caused by increased availability of intracellular [Na+] to the pump, increased affinity of the pump toward intracellular Na+, an increased number of membrane-associated Na+-K+-ATPases, or an increased turnover rate of existing pumps. Activation of pump activity is not inhibited by blockers of the Na+/H+ exchanger (13) and, in this study, occurred in the absence of any observable increase in intracellular [Na+] (Fig. 2). However, when the activity of the Na+-K+-ATPase was inhibited by ouabain, a significant increase in Na+ accumulation was observed (Fig. 3). This increase, however, followed rather than preceded activation of Rb+ uptake by the Na+-K+-ATPase. The delay in the rise of intracellular [Na+] following insulin stimulation was not an artifact of the method used, since an immediate increase in intracellular [Na+] is seen in human fibroblasts stimulated by serum when the same method is used (13). For these reasons, the increase of intracellular [Na+] observed in the presence of ouabain is likely to represent an independent effect of insulin on a separate channel or ion transporter, rather than causing increased Na+-K+-ATPase activity. Increased Na+ influx occurred in the presence of bumetanide and ouabain (Fig. 3), indicating that it is not mediated by the Na+-K+-Cl cotransporter and does not represent a homeostatic mechanism to compensate increased Na+ extrusion by the Na+-K+-ATPase.

Insulin modifies the membrane potential of a number of target cells (23). This is accomplished by modification of ion permeability or stimulation of the Na+-K+-ATPase (12, 24, 35). In human fibroblasts, insulin failed to modify significantly the membrane potential (Fig. 4). While newer methods for the measurement of membrane potential such as the use of microelectrodes or fluorescent dyes could have been more sensitive to transient changes (8), the distribution ratio of arginine used in this study would have detected stable changes in membrane potential induced by insulin. In fact, this system could easily detect changes caused by exposure to ouabain and valinomycin (Fig. 4). These results indicate that the increased sodium extrusion caused by stimulation of the Na+-K+-ATPase was not sufficient to hyperpolarize the cell membrane or that it was compensated by the secondary increase in Na+ accumulation shown in Fig. 3. Changes in membrane potential have been proposed in the past as part of the mechanism of insulin action (23, 34). Our data indicate that stimulation of glucose and amino acid transport in human fibroblasts (14, 17) can well occur in the absence of any change in membrane potential.

Although stimulation of the Na+-K+-ATPase in human fibroblasts occurred in the absence of a significant increase in intracellular Na+, insulin could have increased the affinity of existing pumps to intracellular Na+ (18). Direct measurement of intracellular Na+ indicated that the Na+-K+-ATPase of human fibroblasts had the same affinity toward Na+ in cells stimulated or not stimulated by insulin (Fig. 5). Importantly, at the same concentration of intracellular Na+, the Na+-K+ pump was more active in insulin-treated than in control cells, indicating that insulin increased either the number of active Na+-K+-ATPases or their turnover rate. Measurement of ouabain binding indicated that insulin did not increase the number of membrane-associated Na+-K+-ATPases but slightly increased their affinity toward ouabain (Fig. 6). Because the number of Na+-K+-ATPases was not modified, by exclusion insulin is predicted to stimulate the Na+-K+-ATPase of human fibroblasts by increasing its turnover rate. Previously published data (13) and those in Fig. 6 indicate that insulin increased the turnover rate of the Na+-K+-ATPase of human fibroblasts from 10 to 18.5 cycles/s, assuming that two K+ are taken up by the cell at each cycle (16). The slight, but significant, modification of the Kd toward ouabain induced by insulin is suggestive of possible conformational changes of the Na+-K+-ATPase induced by insulin. Alternatively, since ouabain binds only during part of the Na+-K+-ATPase cycle, insulin may simply increase the rate of a limiting step of pump cycling, increasing both the rate of the overall process and the affinity for ouabain interaction.

After ligand binding, the insulin receptor becomes an active tyrosine kinase and activates a phosphorylation cascade within target cells. This phosphorylation cascade is essential for insulin action on glucose transport. Previous studies in human fibroblasts have shown that genistein, a tyrosine kinase inhibitor (1), and staurosporine, an inhibitor of serine/threonine kinases activated by the insulin receptor (28), block insulin effect on the Na+-K+ pump (13). Additional data presented in this report indicate that insulin stimulation of the Na+-K+-ATPase was not mimicked by the protein kinase C activator PDBU, which inhibited, rather than stimulated, Rb+ transport (Fig. 7). This was relatively unexpected because phorbol esters stimulate glucose transport in human fibroblasts (17). Phorbol esters also stimulate the Na+-K+-ATPase in some cultured skeletal muscle cells (27) but inhibit it in others (2). In the case of the alpha 1-subunit of Na+-K+-ATPase, mutagenesis of the site responsible for protein kinase C phosphorylation results in enzyme activation and prevents further inhibition by phorbol esters (2), indicating that activation of protein kinase C may have an inhibitory action on Na+-K+-ATPase. Our data in fibroblasts, which express the alpha 1-subunit of Na+-K+-ATPase, are consistent with this model and indicate that insulin can reverse the inhibition caused by phorbol esters (Fig. 7). Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, inhibits insulin stimulation of Rb+ transport in 3T3-L1 fibroblasts (30). However, it was without effect in human fibroblasts (Fig. 7), indicating that the mechanism used by insulin to activate Na+-K+-ATPase differs even among similar cell types. This contrasts with insulin activation of glucose transport, in which wortmannin acts as an inhibitor in different cell systems (3, 7), including fibroblasts and Chinese hamster ovary cells (22). The results with kinase activators and inhibitors indicate that insulin stimulates ion and glucose transport with independent mechanisms in human fibroblasts.


    ACKNOWLEDGEMENTS

We thank Dr. Jerry B. Lingrel, University of Cincinnati, Ohio, for kindly providing cDNA probes for the alpha 1, alpha 2, and alpha 3 isoforms of Na+-K+-ATPase.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48742.

Present address of F. Scaglia: Feigin Center, Ste. C235, 6621 Fannin, Department of Molecular and Human Genetics, Baylor College of Medicine, Mail code 3-3370, Houston, TX 77030.

Address for reprint requests and other correspondence: N. Longo, Division of Medical Genetics, Dept. of Pediatrics, Emory Univ., 2040 Ridgewood Drive, Atlanta, GA 30322 (E-mail: nl{at}rw.ped.emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 July 2000; accepted in final form 1 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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