Mentor Sopjani

Regulation of erythrocyte survival by AMP-activated protein kinase

AMP‐activated protein kinase (AMPK), an energy‐sensing enzyme, counteracts energy depletion by stimulation of energy production and limitation of energy utilization. On energy depletion, erythrocytes undergo suicidal death or eryptosis, triggered by an increase in cytosolic Ca2+ activity ([Ca2+]i) and characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. The present study explored whether AMPK participates in the regulation of eryptosis. Western blotting and confocal microscopy disclosed AMPK expression in erythrocytes. [Ca2+]i (Fluo3 fluorescence), cell volume (forward scatter), and PS exposure (annexin V binding) were determined by fluorescence‐activated cell sorting (FACS) analysis. Glucose removal increased [Ca2+]i, decreased cell volume, and increased PS exposure. The AMPK‐inhibitor compound C (20 µM) did not significantly modify eryptosis under glucose‐replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca2+]i by Ca2+ ionophore ionomycin triggered eryptosis, an effect blunted by the AMPK activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR;1 mM). As compared with erythrocytes from wild‐type littermates (ampk+/+), erythrocytes from AMPKctl‐deficient mice (ampk−/−) were significantly more susceptible to the eryptotic effect of energy depletion. The ampk−/− mice were anemic despite excessive reticulocytosis, and they suffered from severe splenomegaly, again pointing to enhanced erythrocyte turnover. The observations disclose a critical role of AMPK in the survival of circulating erythrocytes.—Föller, M., Sopjani, M., Koka, S., Gu, S., Mahmud, H., Wang, K., Floride, E., Schleicher, E., Schulz, E., Münzel, T., Lang, F. Regulation of erythrocyte survival by AMP‐activated protein kinase. FASEB J. 23, 1072–1080 (2009)

Downregulation of NaPi-IIa and NaPi-IIb Na-coupled phosphate transporters by coexpression of Klotho.

Klotho, a transmembrane protein, protease and hormone has been shown to exert a profound effect on phosphate metabolism. Klotho overexpression lowers and Klotho deficiency increases the plasma phosphate concentration, effects in part attributed to an inhibitory effect of Klotho on the formation of 1,25-dihydroxycholecalciferol (1,25(OH) 2D3), the active form of Vitamin D. Beyond that Klotho has been shown to decrease renal tubular phosphate transport more directly. The influence of Klotho on the plasma phosphate concentration contributes to the profound effect of Klotho on ageing and life span. The present study explored whether Klotho influences the major renal tubular (NaPi-IIa) and the major intestinal (NaPi-IIb) phosphate transporters. For functional analysis NaPi-IIa or NaPi-IIb were expressed in Xenopus oocytes both, without or with additional coexpression of Klotho and electrogenic phosphate transport was estimated from the phosphate-induced current (Ip). According to RT-PCR Klotho is expressed in the murine kidney and intestine. Coexpression of Klotho decreased Ip in both NaPi-IIa- and NaPi-IIb-expressing oocytes. Klotho decreased the maximal Ip without appreciably affecting the concentration required for halfmaximal Ip. Treatment of NaPi-IIa- or NaPi-IIb-expressing oocytes with Klotho protein similarly decreased Ip. In conclusion, Klotho down regulates both, renal (NaPi-IIa) and intestinal (NaPi-IIb) phosphate transporters.

Gold stimulates Ca2+ entry into and subsequent suicidal death of erythrocytes

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.

The Serum and Glucocorticoid Inducible Kinases SGK1-3 Stimulate the Neutral Amino Acid Transporter SLC6A19

The neutral amino acid transporter SLC6A19 (B⁰AT1) plays a decisive role in transport of neutral amino acids in the kidney and intestine. Recently, mutations in SLC6A19 were identified that result in severe neutral aminoaciduria known as Hartnup disorder. SLC6A19 expression and function is controlled by the brush-border angiotensin-converting enzyme 2 (ACE2). Beyond that the mechanisms regulating SLC6A19 function are unknown. The SLC6A19 sequence contains a conserved putative phosphorylation site for the serum and glucocorticoid inducible kinase isoforms SGK1-3, kinases known to regulate a variety of channels and transporters. The present study explored the role of SGK1-3 in the regulation of SLC6A19. As shown by two-electrode voltage clamp in the Xenopus oocyte expression system, leucine-induced currents in SLC6A19 expressing oocytes were activated by the protein kinases SGK1-3. The putative phosphorylation site on the transporter is not essential for SLC6A19 regulation by the kinases. As determined by quantitative immunoassay and electrophysiology, the kinases increase SLC6A19 currents by increasing the cell surface expression of the protein without altering the affinity of the carrier. Following inhibition of carrier insertion into the cell membrane by treatment with brefeldin A (BFA), the leucine-induced current declined significantly slower in Xenopus oocytes expressing SLC6A19 together with SGK1 than in oocytes expressing SLC6A19 alone, a finding pointing to SGK-mediated transporter stabilization in the plasma membrane. Coexpression of ACE2 markedly increased leucine-induced currents in SLC6A19 expressing oocytes that were further enhanced by SGK1-3 kinases. In conclusion, SGK isoforms are novel potent stimulators of SLC6A19 and may thus participate in the regulation of neutral amino acid transport in vivo.

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 (Ig), which was half maximal (KM) at ≈ 650 μM glucose concentration. Coexpression of γR70QAMPK did not affect KM 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 Ig. 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.

Vanadate-Induced Suicidal Erythrocyte Death

Vanadium, a trace element, as vanadate (VO₄^3–) is known to interfere with a wide variety of enzymes including Ca²⁺ ATPase and Na⁺/K⁺ ATPase. VO₄^3– is excreted mainly via the kidney. In renal insufficiency, the impaired VO₄^3– excretion leads to VO₄^3– accumulation in blood.The present study explored the effect of VO₄^3– on eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptotic cells are phagocytosed and thus rapidly cleared from circulating blood. Stimulators of eryptosis include an increase of the cytosolic Ca²⁺ concentration. Erythrocyte Ca²⁺ activity was estimated from Fluo-3 fluorescence, phosphatidylserine exposure from annexin V-binding, and erythrocyte volume from forward scatter in FACS analysis. Exposure of erythrocytes to VO₄^3– increased cytosolic Ca²⁺ concentration, enhanced the percentage of annexin V-binding erythrocytes, decreased erythrocyte forward scatter, and lowered the intracellular ATP concentration. In conclusion, VO₄^3– induces eryptosis at least partially through increase of cytosolic Ca²⁺ concentration, an effect presumably contributing to the development of anemia in chronic renal failure.

Stimulation of eryptosis by cadmium ions.

Cadmium ions are known to trigger apoptosis. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. Cellular mechanisms known to trigger cell membrane phospholipid scrambling include increased cytosolic Ca2+ activity and activation of a sphingomyelinase with formation of ceramide. The present experiments were performed to explore whether cadmium ions (Cd2+) trigger phosphatidylserine exposure of erythrocytes and to possibly identify underlying mechanisms. Phosphatidylserine exposure was estimated from annexin V-binding as determined in fluorescence activated cell sorting (FACS) analysis. Exposure to Cd2+ (≧ 5.5 µM Cd2+) indeed significantly increased annexin V-binding. This effect was paralleled by erythrocyte shrinkage as apparent from the decrease of forward scatter in FACS analysis. According to Fluo3 fluorescence, Cd2+ increased the entry of Ca2+ into erythrocytes. According to antibody binding, Cd2+ did not stimulate the formation of ceramide. In the nominal absence of extracellular Ca2+ and in the presence of cation channel inhibitor amiloride the effects of Cd2+ on erythrocyte phosphatidylserine exposure and forward scatter were blunted. In conclusion, in human erythrocytes Cd2+ stimulates entry of Ca2+, which activates Ca2+-sensitive K+ channels leading to erythrocyte shrinkage and triggers Ca2+-sensitive erythrocyte membrane scrambling leading to phosphatidylserine exposure.

Suicidal death of erythrocytes due to selenium-compounds.

Selenium is an essential element incorporated into selenoproteins. Selenium deficiency may predispose to immune deficiency, mood disorders, and cancer. On the other hand, excessive environmental exposure to selenite may cause a variety of disorders including anemia. At least in theory, the anemia could result from accelerated suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by an increase in the cytosolic Ca2+ concentration and by ceramide. The present experiments explored, whether high concentrations of selenite stimulate eryptosis. According to Fluo3 fluorescence, selenite (≥?200 μg/l sodium selenite) within 48 hours significantly increased the cytosolic Ca2+ concentration in human erythrocytes. According to binding of selective fluorescent antibodies, selenite (≥ 200 μg/l) significantly increased ceramide formation. Annexin V-binding demonstrated that selenite (≥200 μg/l) significantly increased phosphatidylserine exposure of erythrocytes. Forward scatter analysis further revealed that selenite (≥ 200 μg/l) significantly decreased cell volume. In contrast to selenite, selenate failed to trigger eryptosis. In conclusion, selenite triggers suicidal erythrocyte death at least partially by increasing the cytosolic Ca2+ concentration and by stimulating the formation of ceramide. The present study discloses novel cellular effects of this essential nutrient.

Stimulation of electrogenic glucose transport by glycogen synthase kinase 3.

Glycogen synthase kinase 3 GSK3β participates in a wide variety of functions including regulation of glucose metabolism. It is ubiquitously expressed including epithelial tissues. However, whether GSK3β participates in the regulation of epithelial transport is not known. The present study thus explored whether GSK3β influences the Na+-coupled transport of glucose. To this end, SGLT1 was expressed in Xenopus oocytes with or without GSK3β and glucose-induced current (Ig) determined by dual electrode voltage clamp. In Xenopus oocytes expressing SGLT1 but not in water-injected oocytes glucose induced an inwardly directed Ig, which was significantly enhanced by coexpression of GSK3β. According to chemiluminescence and confocal microscopy, GSK3β increased the SGLT1 protein abundance in the oocyte cell membrane. To explore whether GSK3β sensitivity of SGLT1 participates in the regulation of electrogenic intestinal glucose transport, Ussing chamber experiments were performed in intestinal segments from gene-targeted knockin mice with mutated and thus PKB/SGK-resistant GSK3α,β (gsk3KI), in which the serine of the PKB/SGK phosphorylation site was replaced by alanine, and from wild type mice (gsk3WT). The glucose-induced current was significantly larger in gsk3KI than in gsk3WT mice. The present observations reveal a novel function of GSK3, i.e. the stimulation of Na+-coupled glucose transport.

Upregulation of Na-coupled glucose transporter SGLT1 by Tau tubulin kinase 2.

The Tau-tubulin-kinase 2 (TTBK2) is a serine/threonine kinase expressed in various tissues including tumors. Up-regulation of TTBK2 increases resistance of tumor cells against antiangiogenic treatment and confers cell survival. Tumor cell survival critically depends on cellular uptake of glucose, which is partially accomplished by SGLT1 (SLC5A1) mediated Na+-coupled glucose transport. The present study explored whether TTBK2 participates in the regulation of SGLT1 activity. To this end, electrogenic glucose transport was determined in Xenopus oocytes expressing SGLT1 with or without wild-type TTBK2, truncated TTBK2[1-450] or kinase inactive mutants TTBK2-KD and TTBK2-KD[1-450]. TTBK2, but not TTBK2[1-450], TTBK2-KD or TTBK2-KD[1-450], increased membrane carrier protein abundance and electrogenic glucose transport capacity in SGLT1-expressing Xenopus oocytes. Thus TTBK2 is a completely novel regulator of Na+-coupled glucose transport.