Sphingomyelinase dependent apoptosis following ... - Springer Link

Apoptosis (2009) 14:878–889 DOI 10.1007/s10495-009-0364-4

ORIGINAL PAPER

Sphingomyelinase dependent apoptosis following treatment of pancreatic beta-cells with amyloid peptides Aß1-42 or IAPP Ying Zhang Æ Felicia Ranta Æ Cai Tang Æ Ekaterina Shumilina Æ Hasan Mahmud Æ Michael Fo¨ller Æ Susanne Ullrich Æ Hans-Ulrich Ha¨ring Æ Florian Lang

Published online: 2 June 2009 Ó Springer Science+Business Media, LLC 2009

Abstract Amyloid peptides interfere with survival of pancreatic beta-cells. In some cells apoptosis is paralleled by ceramide-dependent alterations of ion channel activity. The purpose of the present study was to elucidate the dependence of amyloid peptides Aß1-42 and islet amyloid polypeptide (IAPP)-induced cell death on ceramide formation and ion channel activity in murine pancreatic islet cells. As disclosed by TUNEL (terminal dUTP nick-end labelling) and cleaved caspase 3 staining, apoptotic cell death was induced by Aß1-42, IAPP and exogenously added C2-ceramide in islet cells from wild type mice. In islet cells from acid sphingomyelinase-deficient mice (ASMKO) Aß1-42 and IAPP but not exogenously added N-acetylD-sphingosine (C2-ceramide, 20 lM) failed to stimulate apoptosis. Immunofluorescent staining revealed a stimulatory effect of Aß1-42 on ceramide formation. According to patch clamp experiments, administration of Aß1-42 and IAPP significantly decreased outwardly rectifying whole cell currents in wild type but not in ASMKO islet cells. C2-ceramide but not inactive di-ceramide (20 lM) mimicked the inhibitory effect on Kv channel current. In conclusion, amyloid peptides induce apoptosis of pancreatic Y. Zhang C. Tang E. Shumilina H. Mahmud M. Fo¨ller F. Lang Department of Physiology, University of Tu¨bingen, Tu¨bingen, Germany F. Ranta S. Ullrich H.-U. Ha¨ring Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical Chemistry, University of Tu¨bingen, Tu¨bingen, Germany F. Lang (&) Physiologisches Institut der Universita¨t Tu¨bingen, Gmelinstr. 5, 72076 Tu¨bingen, Germany e-mail: [email protected]

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islet cells at least in part through activation of acid sphingomyelinase resulting in production of ceramide and subsequent inhibition of ion channel activity. Keywords Ceramide Kv channels Cell death Insulin Amylin

Introduction Pancreatic beta cells produce amylin, the islet amyloid polypeptide (IAPP), a 37 amino acid protein [1]. Amylin is a hormone cosecreted in pancreatic ß cells after a meal and has a complementary action to insulin [2]. Amyloid precursor peptides, such as Aß1-42 fragment, trigger suicidal death in a variety of cells including neurons [3–8], endothelial cells [9], neutrophils [10], erythrocytes [11] and pancreatic beta cells [12, 13]. Compelling evidence suggests that membrane permeable oligomers of amylin may be cytotoxic and responsible for the reduction of beta cell mass during the development of type 2 diabetes mellitus [14–16]. On the one hand, islet amyloid polypeptide has been shown to stimulate ß-cell proliferation [17]. On the other hand, extracellular amyloid peptides have been shown to trigger CD95 (FAS) death receptor signalling [18] and elicit suicidal cell death or apoptosis of pancreatic ß-cells [12, 13, 19–29] fostering the development of diabetes mellitus [1, 30–32]. Accordingly, diabetes develops in hemizygous human amylin transgenic mice [33]. Conversely, suppression of proislet amyloid polypeptide enhances survival of pancreatic islet cells [34]. IAPP may be effective by intracellular amyloidogenesis [35] and may lead to cell membrane disruption [36]. Apoptotic pathways induced by amyloids include the endoplasmic reticulum (ER) stress pathway [1, 19, 20,

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37]. Amyloid peptides may be effective through stimulation of Ca2? entry [10, 38, 39], calpain [7], oxidative stress [3, 4, 6], p38 kinase activation [40], MAP kinase activation [27], c-Jun [41], p53 and p21WAF1/CIP1 [42] and, as shown in other cells, by ceramide formation [5]. Ceramide is generated following cell stress and activation of a wide variety of receptors, which stimulate the acid sphingomyelinase leading to translocation of the enzyme to the extracellular leaflet of the cell membrane [43]. Ceramide is involved in the formation of large membrane platforms, which cluster receptor molecules and recruit intracellular signalling molecules to those receptors [43]. Ceramide-enriched membrane platforms facilitate and amplify the respective signalling including triggering of suicidal cell death [43]. In Jurkat T lymphocytes CD95-induced apoptosis is paralleled by inhibition of Kv1.3 K? channels [44, 45], the cell volume regulatory K? channel of those cells [46]. CD95-triggering leads to tyrosine phosphorylation of the voltage gated K? channel Kv1.3 channel protein [44, 47]. Accordingly, CD95-induced inhibition of Kv1.3 requires the lymphocyte Src-like kinase Lck56 [44, 47]. The inhibitory effect of CD95 triggering is mimicked by the sphingomyelinase product ceramide, which similarly induces apoptosis [47, 48]. The present study explored whether amyloid peptides influence Kv channel activity, ceramide formation and survival of pancreatic beta cells.

Materials and methods Materials Amyloid ß1-42, IAPP, N-Acetyl-D-Sphingosine (C2-ceramide), and Dihydroceramide C2 (Di-C2) were purchased from Sigma–Aldrich (Deisenhofen, Germany). Amyloid b-protein fragment 42-1 was purchased from Bachem California (Torrance, CA). Amyloid ß1-42, C2-ceramide or Di-C2 were dissolved in DMSO at the concentration of 100 lM (Amyloid ß1-42 and Amyloid ß42-1) or 10 mM (C2-ceramide and Di-C2). IAPP was dissolved in water (Ampuwa) at a concentration of 500 lM. All substances were stored at -20°C up to 1 month. Cells were incubated either with solvent solution (as control) or test solution as indicated for each experiment. Islet isolation and islet cell culture Islets were isolated from 4 to 8 month old acid sphingomyelinase knockout (ASM-KO) mice on C57/BL6 genetic background and their wild type littermates (age and sex matched). The aSMase knockout mice [49, 50] were a

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kind gift of Dr Verena Jendrossek (University of Tu¨bingen, Germany) and were originally obtained from Dr R Kolesnick (Sloan Kettering Cancer Memorial Center, NY, USA). For digestion of the exocrine part of the pancreas, 3 ml of collagenase solution (1 mg/ml, Serva, Heidelberg, Germany) was injected into the pancreas in situ through the ductus coledochus. The entire gland was removed and incubated for 10 min at 37°C. Thereafter the islets were isolated from the exocrine tissue by collecting them into fresh medium under a dissection microscope. A single islet cells preparation was obtained after trypsin digestion of islets. As digestion with trypsin may lead to degradation of surface proteins and thus alter the function of cells we washed the cells carefully following trypsin digestion. Cells were then cultured for 24–72 h in RPMI 1640 culture medium with 10% serum and antibiotics as described previously [51]. Measurement of apoptotic cell death For TUNEL (terminal dUTP nick-end labelling) and cleaved caspase 3 staining of mouse islet cell nuclei, the cells were seeded onto poly-L-ornithine (0.001%) coated glass cover slips in RPMI 1640 medium. After 1 day culture, test substances were added either in fresh medium (for 24–48 h incubation) or in culture medium without fetal calf serum (FCS) (for 3 h incubation). After incubation, cells were fixed with 4% paraformaldehyde for 1 h at room temperature. TUNEL staining was performed using the protocol provided by the manufacturer (Roche Diagnostics GmbH, TUNEL kit # 1 684 795). For cleaved caspase 3 staining the cells were fixed for 20 min, permeabilized with 0.2% Triton-X-100, blocked for 45 min with PBS supplemented with 10% serum and incubated overnight with rabbit anti-cleaved caspase 3 (1:500, Cell Signalling). As secondary antibody a polyclonal anti-rabbit IgG-AlexaFluor488 (1:400, 1 h) was used. Nuclei were stained with the DNA marker TOPRO3 (1 lM in PBS) for 1 h at room temperature. After washing with PBS, the cells were analysed under a confocal microscope (409 objective; Leica, Germany) using an excitation wavelength of 488 nm for TUNEL or cleaved caspase 3 and 633 nm for TOPRO3. Whole cell current measurements For patch clamp experiments, islet cells were attached to culture dishes and cultured for 1–3 days before the experiment. The dish was mounted in a bath chamber on the stage of an inverted microscope (IM, Zeiss, Jena, Germany) and the cells were superfused at room temperature with a bath solution containing (in mmol/l): 140 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 5.6 glucose and 10 HEPES, pH 7.4. The patch clamp pipettes (GC150 TF-10,

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Harvard Apparatus, March-Hugstetten, Germany) with a resistance of 3–6 MX were pulled using a DMZ-universal puller (Zeitz, Augsburg, Germany). They were filled with an internal solution containing (in mmol/l): 30 KCl, 95 K?-gluconate, 1 MgCl2, 1.2 NaH2PO4, 4.8 Na2HPO4, 5 Na2ATP, 1 Na3GTP, 5 EGTA, pH 7.2. An EPC9 patch clamp amplifier (Heka Electronic, Lambrecht, Germany) was used for current measurements. After reaching a GigaOhm seal between patch pipette and cell, the cells were perfused with bath solution supplemented with 0.1% BSA (bovine serum albumin) and 100 lM tolbutamide, in order to block KATP channels. Substances or DMSO as control were added as indicated. Ceramide staining Ceramide was detected using anti-ceramide antibodies (Mouse IgM, MID 15B4, Alexis) at a dilution of 1:10 in PBS containing 10% FCS and as second antibody FITC0labelled goat anti-mouse IgG antibody at a dilution of 1:400 (Invitrogen, UK) as described previously [52]. Nuclei were stained with TOPRO3 for 1 h. Statistics Data are presented as mean ± SEM. ANOVA (Dunnets test) for multiple groups and Student’s t-tests were used for statistical analysis. P values \0.05 were accepted to indicate statistical significance.

Results Islet cells from acid sphingomyelinase deficient mice (ASMKO) and their wild type littermates were analyzed to examine whether sphingomyelinase participates in the induction of apoptotic cell death following exposure to amyloid peptide Aß1-42 or IAPP. Apoptosis was detected by staining with two apoptotic markers, TUNEL and cleaved caspase 3. As illustrated in Fig. 1, some apoptosis was observed even in the absence of amyloid peptides. Treatment of isolated islet cells from wild type mice under standard culture conditions with amyloid beta fragment Aß1-42 (2 lM) or IAPP (5 lM) for 48 h significantly increased the percentage of TUNEL positive ß-cells, a finding pointing to stimulation of apoptotic cell death by amyloid peptides (Fig. 1a, b). Amyloid ß1-42 peptide increased the percentage of TUNEL stained cells by 25.2 ± 2.5% and IAPP by 24.7 ± 1.0% (P \ 0.01; n = 7 and 5, respectively). In the absence of serum, a 20-fold lower concentration of amyloid ß1-42 (0.1 lM) significantly increased apoptotic cell death of mouse islet cells (Fig. 1b). The cytosolic staining of cleaved caspase 3 further indicated

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that apoptosis rather then necrosis was induced. In ASMKO islet cells neither amyloid peptide Aß1-42 nor IAPP significantly increased the percentage of TUNEL positive cells (P [ 0.05; n = 7 and 5, respectively; Fig. 1c, d). These observations suggest that both peptides, amyloid ß1-42 and IAPP, induce apoptotic cell death via activation of ASM and increased ceramide production. If the resistance of the islet cells from sphingomyelinase deficient mice was due to impaired formation of ceramide in those mice, application of ceramide itself should be similarly effective in islets from sphingomyelinase-deficient mice and wild type mice. Thus, additional experiments were performed with exogenously added ceramide. Isolated islet cells were treated either with the active N-acetyl-D-sphingosine (C2-ceramide, 40 lM) or with the inactive analogue Di-ceramide (40 lM) for 48 h. The active compound C2 but not the inactive Di-C2 significantly augmented the amount of TUNEL positive cells in both preparations of wild type and of ASM-KO mice (Fig. 2a–d). C2 increased the percentage of TUNEL stained cells by 33.1 ± 5.6% in WT mice and by 43.3 ± 10.0% in ASM-KO mice (P \ 0.001 and 0.01; n = 7 and 4, respectively). These finding together with the observation that amyloid peptide Aß1-42 (2 lM) increased cytosolic ceramide staining within 15 and 25 min (Fig. 3a) indicate that cellular ceramide production induced by amyloid peptides may indeed be responsible for the stimulation of suicidal ß-cell death. As ceramide has previously been shown to inhibit the activity of voltage gated K? channels, which in turn participate in the machinery leading to cell death of Jurkat T lymphocytes, the effects of amyloid peptides on whole cell outward current were examined. The administration of amyloid peptide Aß1-42 (0.1 lM) led to a significant decrease of the whole cell outward current within 15 min from 111.5 ± 11.2 to 71.3 ± 13.2 pA/pF (n = 6, P \ 0.01) and within 25 min to 68.3 ± 13.0 pA/pF (n = 6, P \ 0.001). In contrast, outward currents remained constant in the absence of amyloid peptide (Fig. 3b–d, left and middle graphs). Similar to Aß1-42 IAPP (5 lM) reduced the outward current from 128.8 ± 9.8 to 87.3 ± 10.3 pA/pF (n = 6, P \ 0.001) within 15 min and to 78.1 ± 10.0 pA/pF (n = 6, P \ 0.001, Fig. 3b–d, right graphs) within 25 min. In contrast, the inactive non-amyloidogenic peptide (Amyloid ß42-1, 0.1 lM) did not significantly modify whole cell outward currents (n = 6, results not shown). The effects of amyloid peptides on the outward current were mimicked by active C2-ceramide but not the inactive Di-C2 (Fig. 4a–c). Whole cell outward current was reduced by C2 (20 lM) within 15 min from 118.3 ± 10.0 to 90.9 ± 8.6 pA/pF (n = 6, P \ 0.001) and within 25 min to 82.6 ± 10.5 pA/pF (n = 6, P \ 0.001) (Fig. 4, middle graphs). In contrast, the outward current remained constant

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Fig. 1 Amyloid beta fragment Aß1-42 and IAPP induced apoptosis of pancreatic islet cells depends on acid sphingomyelinase. Wild type islet cells (a and b) and ASMKO (c and d) islet cells were prepared and analysed in parallel as described under ‘‘Materials and methods’’. a and c: Representative images illustrating fluorescent TUNEL (green, upper), cleaved caspase 3 (green, lower) and nuclear TOPRO3 (red) staining of cells cultured for 48 h under control culture condition (left 2 pictures), in the presence of 2 lM amyloid peptide Aß1-42 (middle 2 panels) or of 5 lM IAPP (right 2 panels). b and d: The percentage of TUNEL and cleaved caspase 3 stained cells are expressed as arithmetic means ± SEM of n = 4 independent experiments. Experiments with 0.1 lM amyloid peptide Aß1-42 (n = 4) were performed in the absence of serum. * Indicates significant difference (unpaired t-Test, P \ 0.05), ** indicates significant difference (ANOVA, P \ 0.01), *** indicates significant difference (ANOVA, P \ 0.001)

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Fig. 2 C2-ceramide but not diC2 ceramide induces apoptosis in wild type and ASMKO islet cells. Wild type islet cells (a and b) and ASMKO (c and d) islet cells were prepared and analysed in parallel as described under ‘‘Materials and methods’’. a and c: representative images illustrating fluorescent TUNEL (green, upper), cleaved caspase 3 (green, lower pictures) and nuclear TOPRO3 (red) staining of cells cultured for 48 h under control culture conditions (left) or in the presence of 40 lM C2ceramide (middle) or of 40 lM Di-C2 (right). b and d: The percentage of TUNEL and cleaved caspase 3 stained cells are expressed as arithmetic means ± SEM of n = 4 independent experiments. ** Indicates significant difference (ANOVA, P \ 0.01); *** indicates significant difference (ANOVA, P \ 0.001)

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Fig. 3 Amyloid peptides increase ceramide formation and reduce Kv channel currents in pancreatic islet cells a. Fluorescence microphotograph of ceramide abundance in isolated islet cells prior to (left), 15 min (middle) and 25 min (right) following treatment with amyloid peptide Aß1-42 (2 lM). b. Original tracings of voltage gated K? currents prior to (left), 15 min (middle) and 25 min (right) after addition of solvent solution (left recording, Control), of amyloid peptide Aß1-42 (0.1 lM without addition of serum, middle recording) or of IAPP (5 lM, right recording). Whole cell currents were induced by 200 ms voltage pulses increasing by 20 mV steps from -100 to ?80 mV. c. I–V relations expressed as mean current normalized to the cell capacitance ± SEM of n = 6 experiments prior to (white

diamonds) and 15 min (white squares) and 25 min (black triangles) after exposure of pancreatic islet cells to solvent solution (Control), to amyloid peptide Aß1-42 (0.1 lM, middle) or to IAPP (5 lM, right). d Mean peak current amplitude at ?100 mV normalized to the cell capacitance expressed as arithmetic mean ± SEM of n = 6 independent measurements prior to (white bars), 15 min after (hatched bars) and 25 min after (black bars) exposure of the cells to solvent (left graph), to Aß1-42 (0.1 lM, middle graph) or to IAPP (5 lM, right graph). ** Indicates significant difference from respective value prior to addition of Aß1-42 (P \ 0.01; ANOVA). *** Indicates very significant difference from respective value prior to addition of amyloid or IAPP (P \ 0.001; ANOVA)

in the presence of Di-C2 (113.3 ± 16.9 pA/pF before addition, 116.2 ± 9.6 pA/pF after 15 min and 114.2 ± 6.3 pA/pF after 25 min, n = 5, Fig. 4, right graphs).

In ASM-KO islet cells, amyloid peptide Aß1-42 (0.1 lM) and IAPP did not significantly modify the outward current, which approached 115.3 ± 8.7 pA/pF (n = 4) prior to,

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Fig. 4 Exogenously added C2-ceramide increases Kv channel current in pancreatic islet cells. a. Original tracings of voltage gated K? currents prior to (left), 15 min (middle) and 25 min (right) after addition of solvent solution (left recording, Control), of C2-ceramide (20 lM, middle recording) and of Di-C2 (20 lM, right recording). Whole cell currents were induced by 200 ms voltage pulses increasing by 20 mV steps from -100 to ?80 mV. b. I–V relations expressed as mean current normalized to the cell capacitance ± SEM of n = 6 experiments prior to (white diamonds) and 15 min (white squares) and 25 min (black triangles) after exposure of pancreatic

islets cells to solvent (Control, left), to C2-ceramide (20 lM, middle) or to Di-C2 (20 lM, right). c Mean peak current amplitude at ?100 mV normalized to the cell capacitance expressed as arithmetic mean ± SEM of n = 6 independent measurements prior to (white bars), 15 min after (hatched bars) and 25 min after (black bars) exposure of the cells to solvent (left graph), to C2-ceramide (20 lM, middle graph) or to Di-C2-ceramide (20 lM, right graph). *** Indicates very significant difference from respective value prior to addition of C2 (P \ 0.001; ANOVA)

128.9 ± 18.5 (pA/pF, n = 4, P = 0.30) following a 15 min exposure to amyloid peptide Aß1-42 and 130.4 ± 18.5 (pA/pF, n = 4, P = 0.35) after a 25 min exposure to amyloid peptide Aß1-42 (Fig. 5a–c, middle graphs). Likewise, IAPP (5 lM) did not affect the outward current in ASMKO islet cells. The peak current density before addition of IAPP was 120.5 ± 5.9 pA/pF and remained after 15 min perfusion with IAPP at 136.8 ± 10.3 pA/pF and after 25 min at 139.1 ± 11.1 pA/pF (n = 6, Fig. 5, right graphs). In contrast to amyloid peptides, exogenously added ceramide significantly reduced the outward current in the ASM-KO islet cells from 119.7 ± 13.3 to 84.5 ± 9.0 pA/pF (n = 5, P \ 0.05) within 15 min and to 62.3 ± 11.9 pA/pF (n = 5, P \ 0.05) within 25 min (Fig. 5d–f). These observations suggest that both IAPP and amyloid Aß1-42 induce apoptotic cell death and decrease channel

activity in insulin secreting cells at least in part by the stimulation of acid sphingomyelinase and ceramide production.

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Discussion The present study reveals that the beta amyloid fragment 1-42 (Aß1-42) triggers the suicidal death of pancreatic beta cells. The effect of Aß1-42 is obviously secondary to stimulation of acid sphingomyelinase and subsequent formation of ceramide. Accordingly, the effect is mimicked by addition of ceramide. More importantly, genetic knockout of acid sphingomyelinase abrogates the proapoptotic effect of Aß1-42. Even without amyloid peptides and ceramide, some apoptosis is observed, which may have

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resulted from exposure to poly-L-ornithine. The signalling of this basal apoptosis is not clear, it is not paralleled by appreciable ceramide formation. However, the addition of

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Aß1-42, IAPP or ceramide clearly enhanced the apoptosis, an observation underscoring the proapoptotic effect of amyloid peptides and ceramide.

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886 5 The effect of amyloid peptides on Kv channels is abrogated in the absence of ASM. a Original tracings of voltage gated K? currents prior to (left), 15 min (middle) and 25 min (right) after addition of solvent solution (left recording, Control), of amyloid peptide Aß1-42 (0.1 lM, middle recording) or of IAPP (5 lM, right recording). Whole cell currents were induced by 200 ms voltage pulses increasing by 20 mV steps from -100 to ?80 mV. b I–V relations expressed as mean current normalized to the cell capacitance ± SEM of n = 6 experiments prior to (white diamonds) and 15 min (white squares) or 25 min (black triangles) after exposure of pancreatic islets cells to solvent (control, left), to amyloid peptide Aß1-42 (0.1 lM, middle) or to IAPP (5 lM, right). c Mean peak current amplitude at ?100 mV normalized to the cell capacitance expressed as arithmetic mean ± SEM of n = 6 independent measurements prior to (white bars), 15 min after (hatched bars) and 25 min after (black bars) exposure of the cells to solvent (left graph), to amyloid peptide Aß1-42 (0.1 lM, middle graph) or to IAPP (5 lM, right graph). d Original tracings of voltage gated K? currents prior to (left), 15 min (middle) and 25 min (right) after addition of solvent (left recording, control) or of C2-ceramide (20 lM, right recording). Whole cell currents were induced by 200 ms voltage pulses increasing by 20 mV steps from -100 to ?80 mV. e I–V relations expressed as mean current normalized to the cell capacitance ± SEM of n = 6 experiments prior to (white diamonds) and 15 min (white squares) and 25 min (black triangles) after exposure of pancreatic islets cells to solvent (control, left) or to C2-ceramide (20 lM, right). f Mean peak current amplitude at ?100 mV normalized to the cell capacitance expressed as arithmetic mean ± SEM of n = 6 independent measurements prior to (white bars), 15 min after (hatched bars) and 25 min after (black bars) exposure of the cells to solvent (left graph) or to C2-ceramide (20 lM, right graph). * Indicates significant difference from respective value prior to addition of C2-ceramide (P \ 0.05; ANOVA)

b Fig.

Ceramide has been shown to participate in the triggering of cell death in a variety of cells including T-lymphocytes [47], hepatocytes [53], erythrocytes [54–56] and pancreatic beta cells [57]. Aß1-42 further inhibits Kv channel activity in pancreatic beta cells. Apparently, amyloid is again at least partially effective through stimulation of ceramide formation. Ceramide has been shown to modify the activity of a variety of ion channels including K? channels [47, 58–64], Ca2? channels [65, 66] and Na? channels [67]. In some cells inhibition of Kv channels has been shown to compromise cell proliferation [68, 69] and facilitate apoptosis [68, 70–74]. On the other hand, [75, 76] amyloidand ceramide-induced [77] apoptotic cell death may be paralleled by activation of K? channels. Activation of K? channels leads to cell shrinkage, a hallmark of suicidal cell death [78–82]. In pancreatic beta cells Kv channels may further participate in the regulation of insulin release [51, 83]. Accordingly, inhibition of Kv channels augments glucose induced-insulin secretion [83]. Conversely, glucocorticoids stimulate Kv1.5 channels, thus accelerating the repolarisation of the cell membrane upon glucose-induced depolarization and blunting the Ca2? entry through voltage gated Ca2? channels [51].

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The role of ceramide formation in the untoward effects of amyloid peptides may be therapeutically relevant. The sphingomyelinase may be inhibited by several substances including amitriptylin, which indeed favourably influenced the clinical course of Wilson0 s disease in an animal model, a condition caused by excessive ceramide formation [53]. Alternatively, the effect of amyloid peptides may be counteracted by rosiglitazone, an activator of peroxisome proliferator-activated receptor-gamma, which is effective through phosphatidylinositol 30 -kinase-dependent signalling [84]. In conclusion, the toxic amyloid fragment Aß1-42 decreased Kv channel activity and triggered apoptosis of pancreatic beta cells, effects at least in part due to formation of ceramide. The events may contribute to the pathophysiology of amyloid-related disease. Acknowledgments The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic and Sari Ru¨be. This study was supported by the Deutsche Forschungsgemeinschaft (La 315/6-1, La 315/13-1 and GRK1302/1). The authors also thank Daniel Brucker, Hertie Institute for Clinical Brain Research and Laboratory of Molecular Neurooncology, for his kind support. Conflict of interest interest.

The authors declare that there is no conflict of

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