Regulation of the epithelial Na+ channel by intracellular Na+

The hypothesis that the intracellularNa⁺ concentration([Na⁺]_i)is a regulator of the epithelialNa⁺ channel (ENaC) was tested withthe Xenopus oocyte expression systemby utilizing a dual-electrode voltage clamp.[Na⁺]_iaveraged 48.1 ± 2.2 meq (n = 27)and was estimated from the amiloride-sensitive reversal potential.[Na⁺]_iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 MNa₂SO₄.Within minutes of injection,[Na⁺]_istabilized and remained elevated at 97.8 ± 6.5 meq(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after theinitial injection of 0.5 and 0.25 MNa₂SO₄,respectively. This increase of[Na⁺]_icaused a biphasic inhibition of ENaC currents. In oocytes injected with0.5 MNa₂SO₄(n = 9), a rapid decrease of inwardamiloride-sensitive slope conductance(g_Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slowerdecrease to 0.304 ± 0.043 of control at 30 min were observed.Similar but smaller inhibitions were also observed with the injectionof 0.25 MNa₂SO₄.Injection of isotonicK₂SO₄(70 mM) or isotonicK₂SO₄made hypertonic with sucrose (70 mMK₂SO₄-1.2M sucrose) was without effect. Injection of a 0.5 M concentration ofeitherK₂SO₄,N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thusincreases of[Na⁺]_ihave multiple specific inhibitory effects on ENaC that can betemporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

     

Regulation of the epithelial Na+ channel by cytosolic ATP

The epithelial Na+ channel (ENaC), composed of three subunits (alphabetagamma), is expressed in various Na(+)-absorbing epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. By using patch clamp techniques, we have examined the effect of cytosolic ATP on the activity of the rat alphabetagammaENaC (rENaC) stably expressed in NIH-3T3 cells and in Madin-Darby canine kidney epithelial cells. The inward whole-cell current attributable to rENaC activity ran down when these cells were dialyzed with an ATP-free pipette solution in the conventional whole-cell voltage-clamping technique. This run down was prevented by 2 mM ATP (but not by AMP or ADP) in the pipette solution or by the poorly or non-hydrolyzable analogues of ATP (adenosine 5'-O-(thiotriphosphate) and adenosine 5'-(beta,gamma-imino)triphosphate) in both cell lines, suggesting that protection from run down was mediated through non-hydrolytic nucleotide binding. Accordingly, we demonstrate binding of ATP (but not AMP) to alpharENaC expressed in Madin-Darby canine kidney cells, which was inhibited upon mutation of the two putative nucleotide-binding motifs of alpharENaC. Single channel analyses indicated that the run down of currents observed in the whole-cell recording was attributable to run down of channel activity, defined as NPo (the product of the number of channels and open probability). We propose that this novel ATP regulation of ENaC may be, at least in part, involved in the fine-tuning of ENaC activity under physiologic and pathophysiologic conditions.     

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and G?6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.     

Protein kinase regulation of a cloned epithelial Na+ channel

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC- expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of - 100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel''s open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma- rENaC is not activated by PKA.     

Differential effects of protein kinase C on the levels of epithelial Na+ channel subunit proteins

Regulation of epithelial Na(+) channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both beta and gamma, but not alphaENaC. Temporal regulation of beta and gammaENaC by PKC differed; gammaENaC decreased with a time constant of 3.7 +/- 1.0 h, whereas betaENaC decreased in 13.9 +/- 3. 0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na(+) reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both beta and gammaENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na(+) transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of beta and gamma but not alphaENaC protein. These results imply a PKC-dependent, long term decrease in Na(+) reabsorption.     

Regulation of type IIalpha phosphatidylinositol phosphate kinase localisation by the protein kinase CK2.

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIalpha PIP kinase at a single site unique to that isoform - Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

Regulation of the epithelial Na+ channel by the RH domain of G protein-coupled receptor kinase, GRK2, and Galphaq/11

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na(+) absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant ((K220R)GRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 ((D110A)GRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Ras couples phosphoinositide 3-OH kinase to the epithelial Na+ channel

Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells approximately 3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPbetaS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110alpha) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.     

Regulation of taurine homeostasis by protein kinase CK2 in mouse fibroblasts

Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume is a critical step in cell proliferation, differentiation and induction of apoptosis. In the present study, we use mouse NIH3T3 fibroblasts and Ehrlich Lettré ascites tumour cells with different CK2 expression levels. Taurine uptake via the Na⁺ dependent transporter TauT and taurine release are increased and reduced, respectively, following pharmacological CK2 inhibition. The effect of CK2 inhibition on TauT involves modulation of transport kinetics, whereas the effect on the taurine release pathway involves reduction in the open-probability of the efflux pathway. Stimulation of PLA₂ activity, exposure to exogenous reactive oxygen species as well as inhibition of protein tyrosine phosphotases (PTP) potentiate the swelling-induced taurine loss. Inhibition of PI3K and PTEN reduces and potentiates swelling-induced taurine release, respectively. Inhibition of CK2 has no effect on PLA₂ activity and ROS production by NADPH oxidase, whereas it lifts the effect of PTEN and PTP inhibition. It is suggested that CK2 regulates the taurine release downstream to known swelling-induced signal transducers including PLA₂, NADPH oxidase and PI3K.     

Regulation of p53 mediated transactivation by the beta-subunit of protein kinase CK2

The growth suppressor protein p53 plays a main part in cellular growth control. Two of its key functions are sequence specific DNA binding and transactivation. Functions of p53 in growth control are regulated at least in part by its interaction with protein kinases. p53 binds to protein kinase CK2, formerly known as casein kinase 2, and it is phosphorylated by this enzyme. CK2 is composed of two regulating beta-subunits and two catalytic alpha- or alpha'-subunits and the interaction with p53 is mediated by the regulatory beta-subunit of CK2. Recently we showed that the beta-subunit could inhibit the sequence specific DNA binding activity of p53 in vitro. Based on this finding, we asked if a coexpression of the beta-subunit of CK2 with p53 in mammalian cells could inhibit the DNA binding activity of p53 in a physiological context. We found that the coexpression of the beta-subunit showed the same inhibitory effect as in the previous assays with purified proteins. Then, we investigated the effects of the coexpression of the beta-subunit of CK2 on the transactivation and transrepression activity of p53. We found that transactivation of the mdm2, p21(WAF1/CIP1) and cyclin G promoter was inhibited in three different cell lines whereas transactivation of the bax promoter was not affected in COS1 cells but down-regulated in MCO1 and SaosS138V21 cells. p53 mediated transrepression of the fos promoter was not influenced by coexpression of the CK2 beta-subunit. Taken together we propose a cell type dependent fine regulation of the p53 transactivation function by the CK2 beta-subunit in vivo, which does not affect p53 mediated transrepression.     

Regulation of cAMP-sensitive colonic epithelial Na+ channel in oocyte expression system

In amphibian epithelia and in cortical collecting duct the antidiuretic peptide arginine-vasopressin (AVP) stimulates activity of epithelial Na+ channels (ENaCs). Generally, the AVP action upon Na+ (re)absorption is believed to be a cAMP/protein-kinase-A mediated mechanism. In the Xenopus oocyte expression system, however, a clear stimulation of ENaC activity by cAMP could not be reproduced with channel subunits cloned from A6 cells or rat colon. We have recently shown that membrane-permeant 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) stimulates activity of a hybrid ENaC in Xenopus oocytes, that consists of an alpha-subunit cloned from guinea-pig colon and the beta- and gamma-subunit originating from rat colon (gpalpharbetagammaENaC). In the present study, we have further investigated the mechanisms by which cpt-cAMP upregulates gpalpharbetagammaENaC activity. Interestingly, we found AVP to stimulate the gpalpharbetagammaENaC in oocytes. Also, treatment with GTP-gamma-S largely activated this channel. In contrast, as a conflicting result, forskolin had no stimulatory effect on the cAMP-sensitive gpalpharbetagammaENaC. Experiments with Brefeldin A (BFA) or nocodazole suggested that only a minor part of cpt-cAMP-induced activation is probably due to an additional translocation of channel proteins into the oocyte membrane. In conclusion, the stimulatory effect of synthetic cpt-cAMP does not seem to be exclusively provided by classical cAMP/PKA-associated transduction mechanisms, i.e., as in A6 cells.     

Indirect activation of the epithelial Na+ channel by trypsin

We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown.     

Regulation of taurine transport systems by protein kinase CK2 in mammalian cells

Maintaining cell volume is critical for cellular function yet shift in cell volume is a prerequisite for mitosis and apoptosis. The ubiquitously and evolutionary conserved serine/threonine kinase CK2 promotes cell survival and suppresses apoptosis. The present review describes how mammalian cells regulate the cellular content of the major cellular organic osmolyte, taurine with emphasis on CK2 mediated regulation of active taurine uptake and volume-sensitive taurine release. Furthermore, we discuss how CK2-mediated regulation of taurine homeostasis is potentially involved in cellular functions such as proliferation and survival.     

Regulation of the renal-specific Na+-K+-2Cl- co-transporter NKCC2 by AMP-activated protein kinase (AMPK)

The renal-specific NKCC2 (Na+-K+-2Cl- co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.     

Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits

Using the Xenopus oocyteexpression system, we examined the mechanisms by which the - and-subunits of an epithelial Na⁺channel (ENaC) regulate -subunit channel activity and the mechanisms by which -subunit truncations cause ENaC activation. Expression of-ENaC alone produced small amiloride-sensitive currents (43 ± 10 nA, n = 7). These currentsincreased >30-fold with the coexpression of - and -ENaC to1,476 ± 254 nA (n = 20).This increase was accompanied by a 3.1- and 2.7-fold increase ofmembrane fluorescence intensity in the animal and vegetal poles of theoocyte, respectively, with use of an antibody directed against the-subunit of ENaC. Truncation of the last 75 amino acids of the-subunit COOH terminus, as found in the original pedigree ofindividuals with Liddle's syndrome, caused a 4.4-fold(n = 17) increase of theamiloride-sensitive currents compared with wild-type -ENaC.This was accompanied by a 35% increase of animal pole membranefluorescence intensity. Injection of a 30-amino acid peptide withsequence identity to the COOH terminus of the human -ENaCsignificantly reduced the amiloride-sensitive currents by 40-50%.These observations suggest a tonic inhibitory role on the channel'sopen probability (P_o) by the COOH terminus of -ENaC. We conclude that the changes of current observed with coexpression of the - and -subunits or those observed with -subunit truncation are likely the result ofchanges of channel density in combination with large changes ofP_o.

     

Regulation of Na+-coupled glucose carrier SGLT1 by AMP-activated protein kinase

Abstract

AMP-activated protein kinase (AMPK), a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilization. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na⁺-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active ^γR70QAMPK (α1β1γ1(R70Q)) alone, the addition of glucose to the extracellular bath generated a current (I_g), which was half maximal (K_M) at ≈ 650 μM glucose concentration. Coexpression of ^γR70QAMPK did not affect K_M but significantly enhanced the maximal current (≈ 1.7 fold). Coexpression of wild type AMPK or the kinase dead ^αK45RAMPK mutant (α1(K45R)β1γ1) did not appreciably affect I_g. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 μM) enhanced the SGLT1 protein abundance in the cell membrane of Caco2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na⁺-coupled glucose transport.