SPAK-Sensitive Regulation of Glucose Transporter SGLT1

The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK is a powerful regulator of ion transport. The study explored whether SPAK similarly regulates nutrient transporters, such as the Na⁺-coupled glucose transporter SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active ^T233ESPAK, WNK-insensitive ^T233ASPAK or catalytically inactive ^D212ASPAK, and electrogenic glucose transport determined by dual-electrode voltage-clamp experiments. Moreover, Ussing chamber was employed to determine the electrogenic glucose transport in intestine from wild-type mice (spak ^wt/wt) and from gene-targeted mice carrying WNK-insensitive SPAK (spak ^tg/tg). In SGLT1-expressing oocytes, but not in water-injected oocytes, the glucose-dependent current (I _g) was significantly decreased following coexpression of wild-type SPAK and ^T233ESPAK, but not by coexpression of ^T233ASPAK or ^D212ASPAK. Kinetic analysis revealed that SPAK decreased maximal I _g without significantly modifying the glucose concentration required for halfmaximal I _g (K _m). According to the chemiluminescence experiments, wild-type SPAK but not ^D212ASPAK decreased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I _g, which was similar in the absence and presence of SPAK, suggesting that SPAK did not accelerate the retrieval of SGLT1 protein from the cell membrane but rather down-regulated carrier insertion into the cell membrane. Intestinal electrogenic glucose transport was significantly lower in spak ^wt/wt than in spak ^tg/tg mice. In conclusion, SPAK is a powerful negative regulator of SGLT1 protein abundance in the cell membrane and thus of electrogenic glucose transport.     

Up-regulation of Na-coupled glucose transporter SGLT1 by caveolin-1

The Na⁺-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (I_g), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal I_g without significantly modifying the glucose concentration required to trigger half maximal I_g (K_M). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I_g, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.     

Regulation of Na(+)-coupled glucose carrier SGLT1 by human papillomavirus 18 E6 protein

Tumor cells utilize preferably glucose for energy production. They accomplish cellular glucose uptake in part through Na⁺-coupled glucose transport mediated by SGLT1 (SLC5A1). This study explored the possibility that the human papillomavirus 18 E6 protein HPV18 E6 (E6) participates in the stimulation of SGLT1 activity. E6 is one of the two major oncoproteins of high-risk human papillomaviruses, which are the causative agent for cervical carcinoma. According to Western blotting, SGLT1 is expressed in the HPV18-positive cervical carcinoma cell line HeLa. To explore whether E6 affects SGLT1 activity, SGLT1 was expressed in Xenopus oocytes with and without E6 and electrogenic glucose transport determined by dual electrode voltage clamp. In SGLT1-expressing oocytes, but not in oocytes injected with water or expressing E6 alone, glucose triggered a current (I_g). I_g was significantly increased by coexpression of E6 but not by coexpression of E2. According to chemiluminescence and confocal microscopy, coexpression of E6 significantly increased the SGLT1 protein abundance in the cell membrane. The decay of I_g following inhibition of carrier insertion by Brefeldine A (5 μM) was not significantly affected E6 coexpression. Accrodingly, E6 was not effective by increasing carrier protein stability in the membrane. In conclusion, HPV18 E6 oncoprotein participates in the upregulation of SGLT1.     

Inhibition of the heterotetrameric K+ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

Abstract

The heterotetrameric K⁺-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na⁺ channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant ^αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.     

Upregulation of the Na+-Coupled Phosphate Cotransporters NaPi-IIa and NaPi-IIb by B-RAF

B-RAF, a serine/threonine protein kinase, contributes to signaling of insulin-like growth factor IGF1. Effects of IGF1 include stimulation of proximal renal tubular phosphate transport, accomplished in large part by Na⁺-coupled phosphate cotransporter NaPi-IIa. The related Na⁺-coupled phosphate cotransporter NaPi-IIb accomplishes phosphate transport in intestine and tumor cells. The present study explored whether B-RAF influences protein abundance and/or activity of type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb. cRNA encoding wild-type NaPi-IIa and wild-type NaPi-IIb was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type B-RAF, and electrogenic phosphate transport determined by dual-electrode voltage clamp. NaPi-IIa protein abundance in Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified by chemiluminescence. Moreover, in HEK293 cells, the effect of B-RAF inhibitor PLX-4720 on NaPi-IIa cell surface protein abundance was quantified utilizing biotinylation of cell surface proteins and western blotting. In NaPi-IIa-expressing Xenopus oocytes, but not in oocytes injected with water, addition of phosphate to extracellular bath generated a current (I _P), which was significantly increased following coexpression of B-RAF. According to kinetic analysis, coexpression of B-RAF enhanced the maximal I_P. Coexpression of B-RAF further enhanced NaPi-IIa protein abundance in the Xenopus oocyte cell membrane. Treatment of HEK293 cells for 24 h with PLX-4720 significantly decreased NaPi-IIa cell membrane protein abundance. Coexpression of B-RAF, further significantly increased I_P in NaPi-IIb-expressing Xenopus oocytes. Again, B-RAF coexpression enhanced the maximal I_P. In conclusion, B-RAF is a powerful stimulator of the renal and intestinal type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb, respectively.     

Inhibition of Kir2.1 (KCNJ2) by the AMP-activated protein kinase

The inward rectifier K⁺ channel Kir2.1 participates in the maintenance of the cell membrane potential in a variety of cells including neurons and cardiac myocytes. Mutations of KCNJ2 encoding Kir2.1 underlie the Andersen–Tawil syndrome, a rare disorder clinically characterized by periodic paralysis, cardiac arrhythmia and skeletal abnormalities. The maintenance of the cardiac cell membrane potential is decreased in ischaemia, which is known to stimulate the AMP-activated serine/threonine protein kinase (AMPK). This energy-sensing kinase stimulates energy production and limits energy utilization. The present study explored whether AMPK regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant ^αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. Kir2.1 activity was determined in two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Expression of wild type Nedd4-2 or of Nedd4-2^S795A lacking an AMPK phosphorylation consensus sequence downregulated Kir2.1 currents. The effect of wild type Nedd4-2 but not of Nedd4-2^S795A was significantly augmented by additional coexpression of AMPK. In conclusion, AMPK is a potent regulator of Kir2.1. AMPK is at least partially effective through phosphorylation of the ubiquitin ligase Nedd4-2.     

Stimulation of the glucose carrier SGLT1 by JAK2

JAK2 (Janus kinase-2) overactivity contributes to survival of tumor cells and the ^V617FJAK2 mutant is found in the majority of myeloproliferative diseases. Tumor cell survival depends on availability of glucose. Concentrative cellular glucose uptake is accomplished by Na⁺ coupled glucose transport through SGLT1 (SLC5A1), which may operate against a chemical glucose gradient and may thus be effective even at low extracellular glucose concentrations. The present study thus explored whether JAK2 activates SGLT1. To this end, SGLT1 was expressed in Xenopus oocytes with or without wild type JAK2, ^V617FJAK2 or inactive ^K882EJAK2 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of glucose to the extracellular bath generated a current (I_g), which was significantly increased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Kinetic analysis revealed that coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The stimulating effect of JAK2 expression was abrogated by preincubation with the JAK2 inhibitor AG490. Chemiluminescence analysis revealed that JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I_g during inhibition of carrier insertion by brefeldin A was similar in the absence and presence of JAK2. Thus, JAK2 fosters insertion rather than inhibiting retrieval of carrier protein into the cell membrane. In conclusion, JAK2 upregulates SGLT1 activity which may play a role in the effect of JAK2 during ischemia and malignancy.     

Downregulation of the osmolyte transporters SMIT and BGT1 by AMP-activated protein kinase

The myoinositol transporter SMIT (SLC5A3) and the betaine/γ-aminobutyric acid (GABA) transporter BGT1 (SLC6A12) accomplish cellular accumulation of organic osmolytes and thus contribute to cell volume regulation. Challenges of cell volume constancy include energy depletion, which compromises the function of the Na(+)/K(+) ATPase leading to cellular Na(+) accumulation and subsequent cell swelling. Energy depletion is sensed by AMP-activated protein kinase (AMPK). The present study explored whether AMPK influences the activity of SMIT and BGT1. To this end, cRNA encoding SMIT or BGT1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1+AMPKβ1+AMPKγ1), of constitutively active (γR70Q)AMPK (AMPKα1+AMPKβ1+(R70Q)AMPKγ1) or of catalytically inactive (αK45R)AMPK ((K45R)AMPKα1+AMPKβ1+AMPKγ1). Substrate-induced current in dual electrode voltage-clamp experiments was taken as measure of osmolyte transport. As a result, in SMIT-expressing, but not in water-injected Xenopus oocytes, myoinositol, added to the extracellular bath, generated a current (I(SMIT)), which was half maximal (K(M)) at ≈7.2μM myoinositol concentration. Furthermore, in BGT1-expressing, but not in water-injected Xenopus oocytes, GABA added to the bath generated a current (I(GABA)), which was half maximal (K(M)) at ≈0.5mM GABA concentration. Coexpression of AMPK and of (γR70Q)AMPK but not of (αK45R)AMPK significantly decreased I(SMIT) and I(GABA). AMPK decreased the respective maximal currents without significantly modifying the respective K(M). In conclusion, the AMP-activated kinase AMPK is a powerful regulator of the organic osmolyte transporters SMIT and BGT1 and thus interacts with cell volume regulation.     

Downregulation of the Creatine Transporter SLC6A8 by JAK2

Janus-activated kinase-2 (JAK2) participates in the regulation of the Na⁺-coupled glucose transporter SGLT1 and the Na⁺-coupled amino acid transporter SLC6A19. Concentrative cellular creatine uptake is similarly accomplished by Na⁺-coupled transport. The carrier involved is SLC6A8 (CreaT). The present study thus explored whether JAK2 regulates the activity of SLC6A8. To this end, cRNA encoding SLC6A8 was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, constitutively active ^V617FJAK2 or inactive ^K882EJAK2. Electrogenic creatine transport was determined in those oocytes by dual-electrode voltage-clamp experiments. In oocytes injected with cRNA encoding SLC6A8 but not in oocytes injected with water or with cRNA encoding JAK2 alone, addition of 1 mM creatine to the extracellular bath generated an inward current (I _crea). In SLC6A8 expressing oocytes I _crea was significantly decreased by coexpression of JAK2 or ^V617FJAK2 but not by coexpression of ^K882EJAK2. According to kinetic analysis, coexpression of JAK2 decreased the maximal transport rate without significantly modifying the affinity of the carrier. In oocytes expressing SLC6A8 and ^V617FJAK2 I _crea was gradually increased by the JAK2 inhibitor AG490 (40 μM). In SLC6A8 and JAK2 coexpressing oocytes the decline of I _crea following disruption of carrier insertion with brefeldin A (5 μM) was similar in the absence and presence of JAK2. In conclusion, JAK2 is a novel regulator of the creatine transporter SLC6A8, which downregulates the carrier, presumably by interference with carrier protein insertion into the cell membrane.     

Inhibition of Connexin 26 by the AMP-Activated Protein Kinase

Connexins provide intercellular connections that allow passage of ions and small organic molecules. They clamp the cell membrane potential and cellular ion composition to that of neighboring cells. The cell membrane potential and ion composition of an energy-depleted cell could thus be maintained despite its compromised Na⁺/K⁺ activity. By the same token, however, the breakdown of ion gradients in that cell imposes an additional challenge to the neighboring cells, which may jeopardize their survival. Thus, timely closure of connexins may be critically important for the survival of those cells. Energy depletion stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase that senses energy depletion and stimulates several cellular mechanisms to enhance energy production and to limit energy utilization. The present study explored whether AMPK regulates connexin 26. To this end, cRNA encoding connexin 26 was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (α1β1γ1), of the constitutively active ^γR70QAMPK (α1β1γ1[R70Q]) or of the inactive mutant ^αK45RAMPK (α1[K45R]β1γ1). Connexin 26 activity was determined in dual-electrode voltage-clamp experiments. Moreover, connexin 26 abundance was determined in the oocyte cell membrane by chemiluminescence and confocal microscopy. As a result, connexin 26-mediated current and connexin 26 protein abundance were significantly decreased by coexpression of ^γR70QAMPK and, to a lower extent, by wild-type AMPK but not by ^αK45RAMPK. In conclusion, AMPK is a potent regulator of connexin 26.     

Upregulation of Na/H exchanger by the AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O₂ deficiency glycolytic degradation of glucose leads to formation of lactate and H⁺, thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H⁺ export. The present study explored whether AMPK influences Na⁺/H⁺ exchanger (NHE) activity and/or Na⁺-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH_i) was estimated utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺-dependent re-alkalinization (ΔpH_i) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH_i and ΔpH_i in those cells were significantly increased by treatment with AMPK stimulator AICAR (1 mM) and significantly decreased by AMPK inhibitor compound C (10 μM). The effect of AICAR on pH_i and ΔpH_i was blunted in the presence of the Na⁺/H⁺ exchanger inhibitor cariporide (10 μM), but not by the H⁺ ATPase inhibitor bafilomycin (10 nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.     

Inhibition of the heterotetrameric K+ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

The heterotetrameric K(+)-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na(+) channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active (γR70Q)AMPK (α1β1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.     

Regulation of Glucose Transporter SGLT1 by Ubiquitin Ligase Nedd4‐2 and Kinases SGK1, SGK3, and PKB

Objectives : Serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) inhibits the ubiquitin ligase neuronal cell expressed developmentally downregulated 4‐2 (Nedd4‐2), which retards the retrieval of the epithelial Na⁺ channel ENaC. Accordingly, SGK1 enhances ENaC abundance in the cell membrane. The significance of this effect is shown by an association of an E8CC/CT;I6CC polymorphism in the SGK1 gene with increased blood pressure. However, strong expression of SGK1 in enterocytes not expressing ENaC points to further functions of SGK1. This study was performed to test for regulation of Na⁺‐coupled glucose transporter 1 (SGLT1) by Nedd4‐2, SGK1, and/or the related kinases SGK3 and PKB. Additional studies searched for an association of the SGK1 gene with BMI. Research Methods and Procedures : mRNA encoding SGLT1, wild‐type Nedd4‐2, inactive ^C938SNedd4‐2, wild type SGK1, constitutively active ^S422DSGK1 or inactive ^K127NSGK1, wild‐type SGK3, and constitutively active ^T308DS473DPKB or inactive ^T308AS473APKB were injected into Xenopus oocytes, and glucose transport was quantified from glucose‐induced current (I_glc). BMI was determined in individuals with or without the E8CC/CT;I6CC polymorphism. Results: I_glc was significantly decreased by coexpression of Nedd4‐2 but not of ^C938SNedd4‐2. Coexpression of SGK1, ^S422DSGK1, SGK3, or ^T308DS473DPKB, but not of ^K127NSGK1 or ^T308AS473APKB, enhanced I_glc and reversed the effect of Nedd4‐2. SGK1 and SGK3 phosphorylated Nedd4‐2. Deletion of the SGK/PKB phosphorylation sites in Nedd4‐2 blunted the kinase effects. BMI was significantly (p < 0.008) greater in individuals with the E8CC/CT;I6CC polymorphism than in individuals without. Discussion : Overactivity of SGK1 may lead not only to excessive ENaC activity and hypertension but also to enhanced SGLT1 activity and obesity.     

Regulation of the Na+-coupled glutamate transporter EAAT3 by PIKfyve

The Na⁺,glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end, EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I_glu) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I_glu. Coexpression of either, SGK1 or PIKfyve, significantly enhanced I_glu in EAAT3 expressing oocytes. The increased I_glu was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I_glu and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressing EAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant ^K127NSGK1 did not significantly alter I_glu in EAAT3 expressing oocytes and completely reversed the stimulating effect of PIKfyve coexpression on I_glu. The stimulating effect of PIKfyve on I_glu was abolished by replacement of the serine by alanine in the SGK consensus sequence (^S318APIKfyve). Moreover, additional coexpression of ^S318APIKfyve significantly blunted I_glu in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.     

Segment-specific effects of resveratrol on porcine small intestinal dipeptide absorption depend on the mucosal pH and are due to different mechanisms: potential roles of different transport proteins and protein kinases

Numerous beneficial features of the polyphenol resveratrol (RSV) have been demonstrated in several tissues and cell culture models. There is also evidence, that RSV impairs intestinal nutrient transport but the underlying mechanisms are not understood. The aim of the present study was to evaluate whether RSV has also an impact on the H⁺-coupled transport of peptides via the peptide transporter 1 (PepT1) and to characterize RSV mediated changes in the apical abundance of nutrients transport proteins and protein kinases that may be involved.RSV decreased the H⁺-coupled transport of peptides in the porcine small intestines in a pH and location specific manner (jejunum vs ileum) as measured in Ussing chamber experiments. The comparison of the effects of RSV with the effects of the cAMP/PKA-activating agent forskolin indicates that different mechanisms may be responsible in the intestinal segments. Additionally, it seems that the transport of peptides and glucose in the jejunum are inhibited via the same mechanism while there might be two mechanisms involved in the ileum.Functional data and protein expression data indicate, that, besides PepT1, the activity of the Na⁺/H⁺-exchanger 3 (NHE3) may be involved. Protein kinase A (PKA) and AMP-activated kinase (AMPK) are both activated by RSV while the extracellular regulated kinase (ERK) and the serum and glucocorticoid induced kinase (SGK) are widely unaffected. Although PKA and AMPK are activated, AMPK seems not to be related to the effects of RSV. Additionally, both the functional data and the protein expression data reveal some interesting pH- and segment-specific differences.     

Missense mutations in SGLT1 cause glucose–galactose malabsorption by trafficking defects

Glucose–galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na⁺/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [¹⁴C]α-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.     

Involvement of store-operated Ca2 + entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells

G_q/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M₁, M₃ and M₅ subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca^2 +/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca^2 + required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca^2 + entry (SOCE) in AMPK activation by pharmacologically defined M₃ mAChRs in human SH-SY5Y neuroblastoma cells. In Ca^2 +-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2 min. Conversely, in the presence of extracellular Ca^2 + CCh-induced AMPK phosphorylation lasted for at least 180 min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50 μM) that suppressed CCh-induced intracellular Ca^2 + ([Ca^2 +]_i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca^2 + sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca^2 +]_i plateau and AMPK activation. M₃ mAChRs increased glucose uptake and this response required extracellular Ca^2 + and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M₃ mAChRs and suggests that SOCE is a critical process connecting M₃ mAChRs to the control of neuronal energy metabolism.