[Show abstract][Hide abstract] ABSTRACT: Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants.
Nature medicine 06/2013; 19(7). DOI:10.1038/nm.3214 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. Methods: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive (T308A/S473A)PKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant (S318A)PIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. Results: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (I(le)), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of (T308A/S473A)PKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of (S318A)PIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of I(le) following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells.
[Show abstract][Hide abstract] ABSTRACT: Gum Arabic (GA), a nutrient from dried exudate of Acacia senegal, is widely used as emulsifier and stabilizer. It stimulates sodium and water absorption in diarrhea. This study explored the effects of GA in colonic tissue. Mice were treated with GA (10% wt/vol) in drinking water and gene array was performed. As GA modified several tumor-relevant genes, chemical cancerogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by 3 cycles of 3% dextrane sodium sulphate in drinking water) was induced with or without GA treatment. Within 4 days, GA treatment decreased the colonic transcript levels of the angiogenetic factors angiogenin 1, angiogenin 3, and angiogenin 4 by 78 +/- 18%, 88 +/- 15%, and 92 +/- 13%, respectively (n = 5 each), and of further genes including CD38 antigen, aquaporin4, interleukin18, vav-3-oncogene, gamma(+)-amino acid transporter, sulfatase1, ubiquitinD, and chemokine ligand5. According to Western blotting, GA treatment similarly decreased angiogenin protein expression, and according to immunohistochemistry, it decreased ss-catenin expression. Chemical cancerogenesis resulted in multiple colonic tumors within 12 wk. GA treatment (10% wt/wt) in drinking water significantly decreased the number of tumors by 70%. The observations disclose a powerful anticarcinogenic effect of GA. The nutrient could thus be used for the prophylaxis against colon carcinoma particularly in individuals at enhanced risk.
Nutrition and Cancer 07/2010; 62(6):802-10. DOI:10.1080/01635581003605920 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The neutral amino acid transporter SLC6A19 (B(0)AT1) 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.
[Show abstract][Hide abstract] ABSTRACT: The excitatory amino-acid transporter EAAT4 (SLC1A6), a Na(+),glutamate cotransporter expressed mainly in Purkinje cells, serves to clear glutamate from the synaptic cleft. EAAT4 activity is stimulated by the serum and glucocorticoid inducible kinase SGK1. SGK1-dependent regulation of the Na(+),glucose transporter SGLT1 (SLC5A1) and the creatine transporter CreaT (SLC6A8) has recently been shown to involve the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments thus explored whether SGK1-dependent EAAT4-regulation similarly involves PIKfyve. In Xenopus oocytes expressing EAAT4, but not in water injected oocytes, glutamate induced a current which was significantly enhanced by coexpression of PIKfyve and SGK1. The glutamate induced current in Xenopus oocytes coexpressing EAAT4 and both, PIKfyve and SGK1, was significantly larger than the current in Xenopus oocytes expressing EAAT4 together with either kinase alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter glutamate induced current in EAAT4-expressing Xenopus oocytes and abolished the stimulation of glutamate induced current by coexpression of PIKfyve. The stimulating effect of PIKfyve was abrogated by replacement of the serine with alanine in the SGK consensus sequence ((S318A)PIKfyve). Furthermore, coexpression of (S318A)PIKfyve significantly blunted the stimulating effect of SGK1 on EAAT4 activity. The observations disclose that PIKfyve indeed participates in the regulation of EAAT4.
[Show abstract][Hide abstract] ABSTRACT: The Na(+),glutamate cotransporter EAAT2 is expressed in astrocytes and clears glutamate from the synaptic cleft. EAAT2 dependent currrent is stimulated by the serum and glucocorticoid inducible kinase SGK1. Phosphorylation targets of SGK1 include the human phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). Nothing is known, however, on the role of PIKfyve in the regulation of EAAT2. The present experiments thus explored, whether PIKfyve expression modifies EAAT2 dependent currrent and protein abundance in the cell membrane. In Xenopus oocytes expressing EAAT2 but not in water injected oocytes application of glutamate (2 mM) induced an inward current (I(glu)). Coexpression of either, SGK1 or PIKfyve, significantly enhanced I(glu) in EAAT2 expressing oocytes. I(glu) was significantly higher in Xenopus oocytes coexpressing EAAT2, SGK1 and PIKfyve than in Xenopus oocytes expressing EAAT2 and either, SGK1 or PIKfyve, alone. Additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase (K127N)SGK1 did not significantly alter I(glu) in EAAT2 expressing oocytes and significantly decreased I(glu) in oocytes coexpressing EAAT2 together with PIKfyve. The stimulating effect of PIKfyve on I(glu) was abrogated by replacement of the serine in the SGK consensus sequence by alanine ((S318A)PIKfyve). Furthermore, additional coexpression of (S318A)PIKfyve virtually abolished I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT2. Confocal microscopy reveals that PIKfyve enhances the EAAT2 protein abundance in the cell membrane. The observations disclose that PIKfyve indeed participates in the regulation of EAAT2.
[Show abstract][Hide abstract] ABSTRACT: 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 SGK1include 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 orPIKfyve, 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 expressingEAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter I(glu) in EAAT3 expressing oocytes and completely reversed the stimulating effect ofPIKfyve 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 ((S318A)PIKfyve). Moreover, additional coexpression of(S318A)PIKfyve significantly blunted I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.
Neurochemistry International 05/2009; 54(5-6):372-7. DOI:10.1016/j.neuint.2009.01.002 · 3.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kv1.5 belongs to the family of voltage-gated potassium (Kv) channels and contains a N- and a C-terminal PDZ-binding motif that might be recognized by PDZ domains on the scaffold proteins NHERF1 and NHERF2. Expression studies in Xenopus oocytes demonstrated that NHERF1 and NHERF2 activate Kv1.5, an effect requiring the C-terminal PDZ-binding motif on Kv1.5. NHERF2 enhances Kv1.5 activity and cell surface expression as determined by electrophysiology and immunoassays. NHERF2 elevates Kv1.5 abundance at the plasma membrane by decreasing channel internalization as proven by Brefeldin A experiments. Kv1.5 is stimulated by the serum and glucocorticoid inducible kinase SGK1, a kinase known to interact with the second PDZ domain of NHERF2. This study aims to identify if SGK1 and NHERF2 synergize to increase Kv1.5 currents. Expression of NHERF2 potentiated SGK1-mediated Kv1.5 activation, which was significantly attenuated by deletion of the second PDZ domain in NHERF2. Specificity of observed effects was verified by evaluating the influence of NHERFs on Kv1.3, a known SGK1 target that contains an internal PDZ binding motif. In summary, our results suggest that NHERFs might participate in the regulation of electrical excitability in part by controlling Kv1.5 surface abundance and by clustering signal transduction molecules to the channel.
[Show abstract][Hide abstract] ABSTRACT: Mechanisms of regulatory cell volume increase following cell shrinkage include accumulation of organic osmolytes such as betaine, taurine, sorbitol, glycerophosphorylcholine (GPC) and myo-inositol. Myo-inositol is taken up by the sodium-myo-inositol-transporter SMIT1 (SLC5A3) expressed in a wide variety of cell types. Hypertonicity induces the transcription of the SMIT1 gene upon binding of the transcription factor tonicity enhancer binding protein (TonEBP) to tonicity responsive enhancers (TonE) in the SMIT1 promoter region. However, little is known about post-translational regulation of the carrier protein. In this study we show that SMIT1 is modulated by the serum- and glucocorticoid-inducible kinase SGK1, a protein genomically up-regulated by hypertonicity. As demonstrated by two-electrode voltage-clamp in the Xenopus oocyte expression system, SMIT1-mediated myo-inositol-induced currents are up-regulated by coexpression of wild type SGK1 and constitutively active (S422D)SGK1 but not by inactive (K127N)SGK1. The increase in SMIT1 activity is due to an elevated cell surface expression of the carrier while its kinetic properties remain unaffected. According to the decay of SMIT1 activity in the presence of brefeldin A, SGK1 stabilizes the SMIT1 protein in the plasma membrane. The SGK isoforms SGK2, SGK3 and the closely related protein kinase B (PKB) are similarly capable of activating SMIT1 activity. SMIT1-mediated currents are decreased by coexpression of the ubiquitin-ligase Nedd4-2, an effect counteracted by additional coexpression of SGK1. In conclusion, the present observations disclose SGK isoforms and protein kinase B as novel regulators of SMIT1 activity.
The Journal of Physiology 04/2008; 586(6):1539-47. DOI:10.1113/jphysiol.2007.146191 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The serum and glucocorticoid inducible kinase SGK1 is involved in dexamethasone-induced inhibition of insulin secretion by increasing voltage-gated potassium channel (Kv) activity. SGK1 upregulates the Kv1.5 channel but the precise mechanism underlying the SGK1 dependent regulation of Kv1.5 has not been defined yet. The present study explored the signal transduction processes involved. Expression studies in Xenopus oocytes revealed that SGK1 promotes channel activity by interfering with the Nedd4-2 ubiquitination pathway, irrespective of the presence of putative SGK1 phosphorylation sites on Kv1.5. Expression of the ubiquitin ligase Nedd4-2 declined Kv1.5 currents by ubiquitinating and thereby reducing Kv1.5 plasma membrane expression. Increasing concentrations of SGK1 gradually compensated the inhibiting effect of Nedd4-2 on Kv1.5. Enhanced Kv1.5 surface abundance by SGK1 reflects decreased channel internalization as indicated by Brefeldin A experiments. In conclusion, Kv1.5 upregulation by SGK1 involves inhibition of channel ubiquitination by Nedd4-2 that leads to Kv1.5 stabilization in the plasma membrane. Our results suggest that the kinase might participate in the regulation of insulin secretion in part by controlling Kv1.5 surface abundance.
[Show abstract][Hide abstract] ABSTRACT: PEPT1 and PEPT2 are members of the family of proton-dependent oligopeptide transporters that mediate electrogenic uphill transport of small peptides and peptidomimetics into a variety of cells. Kinetic properties and substrate recognition sites of those transporters have been well defined previously. Little is known, however, about regulation of those transporters. Both PEPT isoforms contain putative phosphorylation sites for the serum and glucocorticoid inducible kinase SGK1 and a C-terminal PDZ binding motif that might be recognized by PDZ domains of the Na(+)/H(+) exchanger regulatory factors NHERF1 and NHERF2. Thus, the present study attempted to clarify the role of SGK1 and NHERFs in the modulation of PEPT isoforms. Expression studies in Xenopus oocytes with subsequent electrophysiology and immunoassays revealed that SGK1 and NHERF2, but not the NHERF1 isoform specifically enhance PEPT2 function and surface abundance. The kinase is effective through phosphorylation of (185)Ser within the SGK1 consensus site, since disruption of this site prevented transporter modulation by the kinase. NHERF2 failed to regulate the C-terminal deletion mutant (PEPT2DeltaC) indicating that the C-terminal PDZ-binding motif in PEPT2 governs transport modulation by NHERF2. Coexpression of NHE3 stimulates PEPT2 activity to a similar extent as coexpression of NHERF2. Dynasore experiments demonstrated that SGK1 and NHERF2 activate PEPT2 by stabilizing the transporter at the cell surface. In conclusion, the present results reveal two novel PEPT2 posttranslational modulators, SGK1 and NHERF2, which might regulate transport of oligopeptides and peptidomimetic drugs.
[Show abstract][Hide abstract] ABSTRACT: Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Using cDNA microarray analysis, we identified SGK1 (serum- and glucocorticoid-regulated kinase 1) as a gene aberrantly expressed in midsecretory endometrium of women with unexplained infertility. SGK1 is a serine/threonine kinase involved primarily in epithelial ion transport and cell survival responses. Real-time quantitative PCR analysis of a larger, independent sample set timed to coincide with the period of uterine receptivity confirmed increased expression of SGK1 transcripts in infertile women compared with fertile controls. We further demonstrate that SGK1 expression is regulated by progesterone in human endometrium in vivo as well as in explant cultures. During the midsecretory phase of the cycle, SGK1 mRNA and protein were predominantly but not exclusively expressed in the luminal epithelium, and expression in this cellular compartment was higher in infertile women. In the stromal compartment, SGK1 expression was largely confined to decidualizing cells adjacent to the luminal epithelium. In primary culture, SGK1 was induced and phosphorylated upon decidualization of endometrial stromal cells in response to 8-bromo-cAMP and progestin treatment. Moreover, overexpression of SGK1 in decidualizing cells enhanced phosphorylation and cytoplasmic translocation of the forkhead transcription factor FOXO1 and inhibited the expression of PRL, a major decidual marker gene. Conversely, knockdown of endogenous SGK1 by small interfering RNA increased nuclear FOXO1 levels and enhanced PRL expression. The observation that SGK1 targets FOXO1 in differentiating human endometrium, together with its distinct temporal and spatial expression pattern and increased expression in infertile patients, suggest a major role for this kinase in early pregnancy events.
[Show abstract][Hide abstract] ABSTRACT: EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [(3)H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ((T40A)EAAT4) or of both phosphorylation sites ((T40AT504A)EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the (T40AT504A)EAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2.
Journal of Neurochemistry 09/2007; 102(3):858-66. DOI:10.1111/j.1471-4159.2007.04585.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin stimulates glucose transport into muscle and fat cells by enhancing GLUT4 abundance in the plasma membrane through activation of phosphatidylinositol 3-kinase (PI3K). Protein kinase B (PKB) and PKCzeta are known PI3K downstream targets in the regulation of GLUT4. The serum- and glucocorticoid-inducible kinase SGK1 is similarly activated by insulin and capable to regulate cell surface expression of several metabolite transporters. In this study, we evaluated the putative role of SGK1 in the modulation of GLUT4. Coexpression of the kinase along with GLUT4 in Xenopus oocytes stimulated glucose transport. The enhanced GLUT4 activity was paralleled by increased transporter abundance in the plasma membrane. Disruption of the SGK1 phosphorylation site on GLUT4 ((S274A)GLUT4) abrogated the stimulating effect of SGK1. In summary, SGK1 promotes glucose transporter membrane abundance via GLUT4 phosphorylation at Ser274. Thus, SGK1 may contribute to the insulin and GLUT4-dependent regulation of cellular glucose uptake.
Biochemical and Biophysical Research Communications 06/2007; 356(3):629-35. DOI:10.1016/j.bbrc.2007.03.029 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mineralocorticoids enhance expression and insulin stimulates activity of the serum- and glucocorticoid-inducible kinase SGK1, which activates the renal epithelial Na+)channel (ENaC). Under a salt-deficient diet, SGK1 knockout mice (sgk1-/-) excrete significantly more NaCl than their wild-type littermates (sgk1+/+) and become hypotensive. The present experiments explored whether SGK1 participates in the hypertensive effects of a high-fat diet and high-salt intake. Renal SGK1 protein abundance of sgk1+/+ mice was significantly elevated after a high-fat diet. Under a control diet, fluid intake, blood pressure, urinary flow rate, and urinary Na+, K+, and Cl- excretion were similar in sgk1-/- and sgk1+/+ mice. Under a standard diet, high salt (1% NaCl in the drinking water for 25 days) increased fluid intake, urinary flow rate, and urinary Na+, K+, and Cl- excretion similarly in sgk1-/- and sgk1+/+ mice without significantly altering blood pressure. A high-fat diet alone (17 wk) did not significantly alter fluid intake, urinary flow rate, urinary Na+, K+, or Cl- excretion, or plasma aldosterone levels but increased plasma insulin, total cholesterol, triglyceride concentrations, and systolic blood pressure to the same extent in both genotypes. Additional salt intake (1% NaCl in the drinking water for 25 days) on top of a high-fat diet did not affect hyperinsulinemia or hyperlipidemia but increased fluid intake, urinary flow rate, and urinary NaCl excretion significantly more in sgk1-/- than in sgk1+/+ mice. Furthermore, in animals receiving a high-fat diet, additional salt intake increased blood pressure only in sgk1+/+ mice (to 132 +/- 3 mmHg) but not in sgk1-/- mice (120 +/- 4 mmHg). Thus lack of SGK1 protects against the hypertensive effects of a combined high-fat/high-salt diet.
American journal of physiology. Renal physiology 01/2007; 291(6):F1264-73. DOI:10.1152/ajprenal.00299.2005 · 3.25 Impact Factor