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AMP-activated protein kinase (AMPK) is an essential cellular energy sensor that senses the cellular energy status and maintains cellular energy balance. The AMPK coordinates cellular and whole-body energy homeostasis through stimulating catabolic ATP-producing and suppressing anabolic ATP-consuming intracellular signaling pathways. AMPK induces autophagy and inhibits cell growth in response to starvation, a process that involves regulating certain intracellular signaling molecules. Recent advances demonstrated the AMPK to exert tumor suppressor activity realized through various signaling molecules by stimulating different cellular processes such as apoptosis, autophagy and cell growth and proliferation. AMPK can also be used to protect against metabolic syndrome. AMPK has previously been reported to be either directly or indirectly involved in the regulation of many different cellular transport proteins of high importance for cellular physiology and pathophysiology. Thus, AMPK provides a necessary link between cellular energy metabolism and cellular transport activities. A better understanding of the AMPK role in intracellular signaling under physiological and pathological conditions may represent a potential strategy for developing therapies for treating many different human diseases and disorders, in which AMPK plays a key role., (© 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Voltage-gated Kv1.5 channels are expressed in a wide variety of tissues including cardiac myocytes, smooth muscle and tumor cells. Kv1.5 channel activity is modified by N-cadherin, which in turn binds the multifunctional oncogenic protein β-catenin. The present experiments explored the effect of β-catenin on Kv1.5 channel activity. To this end, Kv1.5 was expressed in Xenopus oocytes with or without β-catenin and the voltage-gated Kv current determined by dual electrode voltage clamp. As a result, expression of β-catenin significantly increased the voltage-gated Kv current at positive potentials. The stimulating effect of β-catenin on Kv1.5 was not dependent on the stimulation of transcription since it was observed even in the presence of the transcription inhibitor actinomycin D. Specific antibody binding to surface Kv1.5 in Xenopus oocytes revealed that β-catenin enhances the membrane abundance of Kv1.5. Further experiments with brefeldin A showed that β-catenin fosters the insertion of Kv1.5 into rather than delaying the retrieval from the plasma membrane. According to electrophysiological recordings with mutant β-catenin, the effect on Kv1.5 requires the same protein domains that are required for association of β-catenin with cadherin. The experiments disclose a completely novel function of β-catenin, i.e. the regulation of Kv1.5 channel activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.

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 (γR70Q)AMPK (α1β1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

JAK2 (Janus kinase-2) overactivity contributes to survival of tumor cells and the (V617F)JAK2 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, (V617F)JAK2 or inactive (K882E)JAK2 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 (V617F)JAK2, but not by coexpression of (K882E)JAK2. 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

β-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. β-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that β-catenin influences membrane transport. To this end, β-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of β-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na(+)/K(+)-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of β-catenin on the endogenous Na(+)/K(+)-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of β-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of β-catenin expression. The stimulating effect of β-catenin on both Na(+)/K(+) ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of β-catenin, i.e. the regulation of transport., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The suicidal death of erythrocytes, eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. Erythrocyte cell membrane scrambling is stimulated by increase of cytosolic Ca2+ concentration ([Ca2+](i)) and formation of ceramide. Phosphatidylserine (PS) exposing cells are rapidly cleared from circulating blood. Ca2+ entry and/or ceramide formation and thus eryptosis are triggered by lead, mercury, aluminium, and copper ions. The present study explored whether eryptosis could be similarly triggered by exposure to gold. To this end, erythrocytes from healthy volunteers were exposed to AuCl and phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), [Ca2+](i) (Fluo3-dependent fluorescence), and ceramide formation (anti-ceramide-FITC fluorescence) were determined by flow cytometry. Exposure of erythrocytes to low concentrations of AuCl (> or =0.75microg/ml) increased [Ca2+](i) but did not affect ceramide formation. AuCl at concentrations > or =0.5microg/ml significantly increased the number of PS exposing erythrocytes and decreased forward scatter at low concentrations of AuCl pointing to cell shrinkage. Aurothiomalate (> or =1microg/ml), a gold containing drug effective against rheumatoid arthritis, similarly triggered PS exposure of erythrocytes. The present observations disclose a novel action of gold, which may well contribute to side effects during treatment with gold preparations.