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Die Rolle von Serin-Threonin-Kinasen für epitheliale Transportvorgänge
Get PDFThe phosphoinositide dependent kinase PDK1 activates the SGK isoforms SGK1, SGK2 and SGK3 and protein kinase B isoforms which in turn are known to stimulate a variety of sodium coupled transporters, such as the renal and intestinal Na+-dependent glucose transporter SGLT1. SGK1 is known to be up-regulated by mineralocorticoids and to enhance ENaC activity in several expression systems. Moreover, the amiloride-sensitive transepithelial potential difference in collecting duct is lower in gene-targeted mice lacking SGK1 (sgk1-/-) than in their wild type littermates (sgk1+/+). Accordingly, the ability of sgk1-/- mice to decrease urinary sodium output during salt depletion is impaired. ENaC activity and thus transepithelial potential difference in the colon are similarly influenced by mineralocorticoids.
The first aim of the present study was to explore the role of PDK1 in in electrogenic glucose and amino-acid transport in small intestine and proximal renal tubules As mice completely lacking functional PDK1 are not viable, mice expressing 10-25% of PDK1 (pdk1hm) were compared to their wild type littermates (pdk1wt). Body weight was significantly smaller in pdk1hm than in pdk1wt mice. Despite lower body weight of pdk1hm mice, food and water intake were similar in pdk1hm and pdk1wt mice.
Ussing chamber experiments showed that electrogenic transport of glucose as well as phenylalanine, cysteine, glutamine, proline, leucine and tryptophan was significantly smaller in jejunum of pdk1hm mice compared to pdk1wt mice. Similarly, proximal tubular electrogenic glucose transport as well as phenylalanine, glutamine and proline transport in isolated perfused renal tubule segments was decreased. Intraperitoneal injection of 3 g/kg bw glucose resulted in a similar increase of plasma glucose concentration in pdk1hm and in pdk1wt mice but led to a higher increase of urinary glucose excretion in pdk1hm mice. The urinary excretion of proline, valine, guanidinoacetate, methionine, phenylalanine, citrulline, glutamine/glutamate and tryptophan was significantly larger in pdk1hm than in pdk1wt mice. According to immunoblotting of brush border membrane proteins prepared from kidney, expression of the Na+-dependent neutral amino acid transporter B0AT1 (SLC6A19), the glutamate transporter EAAC1/EAAT3 (SLC1A1) and the transporter for cationic amino acids and cystine b0,+AT (SLC7A9) was decreased but the Na+/proline cotransporter SIT (SLC6A20) was increased in pdk1hm mice. In conclusion, reduction of functional PDK1 leads to impairment of intestinal absorption and renal reabsorption of amino acids of electrogenic intestinal glucose absorption and renal glucose reabsorption. The combined intestinal and renal loss of amino acids may contribute to the growth defect of PDK1 deficient mice. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine.
The next step was to look at the transepithelial potential (Vte) and the apparent amiloride-sensitive equivalent short circuit current (Iamil) in colon from sgk1-/- and sgk1+/+ mice. Both Vte and Iamil were significantly (p<0.05) higher in untreated sgk1-/- than in untreated sgk1+/+ mice under control diet. A 7 day exposure to low salt diet increased Vte and Iamil in both genotypes but did not abrogate the differences of Vte and Iamil between sgk1-/- and sgk1+/+ mice. Plasma aldosterone levels were significantly higher in sgk1-/- than in sgk1+/+ mice both under control conditions and under low salt diet. Treatment with dexamethasone (10µg/g BW) or with DOCA (1.5mg per day) significantly increased Vte and Iamil in sgk1+/+ mice but not in sgk1-/- mice. Under treatment with dexamethasone or DOCA Vte and Iamil were similar in sgk1-/- and sgk1+/+ mice. In conclusion, lack of SGK1 does not disrupt colonic ENaC activity and its regulation by salt depletion.
Finally the functional significance of SGK3-dependent regulation of intestinal transport were studied. .Xenopus oocyte coexpression experiments revealed the capacity of SGK3 to up-regulate a variety of transport systems including the sodium-dependent glucose transporter SGLT1. To this end experiments were performed in gene targeted mice lacking functional sgk3 (sgk3-/-) and their wild type littermates (sgk3+/+). Oral food intake and fecal dry weight were significantly larger in sgk3-/- than in sgk3+/+mice. Glucose-induced current (Ig) in Ussing chamber as a measure of Na+ coupled glucose transport was significantly smaller in sgk3-/- than in sgk3+/+mouse jejunal segments. Fasting plasma glucose concentrations were significantly lower in sgk3-/- than in sgk3+/+mice. Intestinal electrogenic transport of phenylalanine, cysteine, glutamine and proline were not significantly different between sgk3-/- and sgk3+/+ mice. In conclusion, SGK3 is required for adequate intestinal Na+ coupled glucose transport and impaired glucose absorption may contribute to delayed growth and decreased plasma glucose concentrations of SGK3 deficient mice. The hypoglycemia might lead to enhanced food intake to compensate for impaired intestinal absorption.Die phosphoinositid-abhängige Kinase 1 (PDK1) aktiviert die SGK-Isoformen SGK1, SGK2 und SGK3 sowie die Isoformen der Proteinkinase B, die eine Vielzahl von Natrium-gekoppelten Transportern stimulieren wie z.B. den renalen und intestinalen glucose-Transporter SGLT1. SGK1 wird durch Mineralokortikoide hochreguliert und stimuliert die Aktivität des epithelialen Natrium-Kanals ENaC in verschiedenen Expressionssystemen. In SGK1 defizienten Mäusen (sgk1-/-) ist die amilorid-hemmbare transepitheliale Potentialdifferenz niedriger als in Wildtyp-Mäusen, unter Niedrigsalz-Diät ist die Fähigkeit zur Na-Konservierung in den sgk1-/- Mäusen eingeschränkt. Die ENaC-Aktivität im Kolon ist ähnlich wie in der Niere mineralokortikoid-abhängig.
Das Ziel der ersten Studie war es, den Einfluss der PDK1 am elektrogenen Glukose- und Aminosäurentransport im Dünndarm und im proximalen Tubulus zu untersuchen. Da Mäuse mit vollständigem PDK1-Verlust nicht lebensfähig sind, wurden PDK1-hypomorphe Mäuse (pdk1hm) mit einer PDK1-Restaktivität von 10-25% untersucht. Das Körpergewicht der PDK1-hypomorphen Mäuse war signifikant geringer als dasjenige der Wildtyp-Tiere, die Futter- und Flüssigkeitsaufnahme waren jedoch ähnlich hoch.
Ussing-Kammer-Experimente zeigten einen reduzierten elektrogenen Transport für Glukose sowie für die Aminosäuren Phenylalanin, Cystein, Glutamin, Prolin, Leucin und Tryptophan in pdk1hm -Mäusen verglichen mit pdk1wt Mäusen. Analog dazu war der elektrogene Transport im isolierten proximalen Tubulus für Glucose sowie für Phenylalanin, Glutamin und Prolin vermindert. Unter intraperitonealer Beladung mit 3 g/kg Glucose kam es zu einer Glukosurie in pdk1hm -Mäusen, trotz ähnlich hoher Glukose-Spiegel wie in Wildtyp-Mäusen. Die Urinausscheidung von Prolin, Valin, Guanidinoacetat, Methionin, Phenylalanin, Citrullin, Glutamine/Glutamat und Tryptophan war in pdk1hm -Mäusen signifikant höher als in Widltyp-Mäusen. Im Western Blot von renalen Bürstensaum-Membranen von pdk1hm Mäusen war die Expression der Na+-abhängigen neutralen Aminosäuren-transporter B0AT1 (SLC6A19), des Glutamat-Transporters EAAC1/EAAT3 (SLC1A1) sowie des Transporter für kationische Aminosäuren und Cystin b0,+AT (SLC7A9) erniedrigt, die Expression des Na+-Prolin Cotransporters SIT (SLC6A20) erhöht. Zusammenfassend konnte gezeigt werden, dass eine Reduktion der PDK1 zu einer verminderten intestinalen Absorption sowie renalen Reabsorption von Glukose und Aminosäuren führte, was auf einen bisher nicht bekannten Regulationsweg hinweist. Der kombinierte intestinale und renale Verlust von Aminosäuren und Glukose könnte zum Minderwuchs der PDK1 hypomorphen Mäuse beitragen.
In weiteren Untersuchungen wurde die transepitheliale Potentialdifferenz (Vte) und der amilorid-hemmbare Kurzschluss-Strom (Iamil) im Kolon von SGK1 defizienten (sgk1-/-) und Wildtyp-Mäusen untersucht. Sowohl Vte und Iamil waren in unbehandelten sgk1-/--Mäusen unter Kontrolldiät signifikant höher als in sgk1+/+ Mäusen. Eine 7-tägige Behandlung mit einer Niedrigsalz-Diät erhöhte Vte und Iamil in beiden Genotypen, konnte den Unterschied in Vte und Iamil zwischen sgk1-/-- und Wildtyp-Mäusen jedoch nicht aufheben. Plasma-Aldosteron-Spiegel waren in sgk1-/- -Mäusen sowohl unter Kontroll- wie Niedrigsalz-Diät signifikant höher. Behandlung mit Dexamethason (10µg/g) oder mit DOCA (1.5mg/ die) erhöhte Vte und Iamil nur in Wildtyp-Mäusen, jedoch nicht in sgk1-/- Mäusen. Unter Behandlung mit Dexamethason oder DOCA waren sowohl Vte als auch Iamil in sgk1-/- und Wildtyp-Mäusen ähnlich hoch. Die Ergebnisse zeigen zusammengenommen, dass ein Fehlen der SGK1 nicht die Aktivität und Regulation des ENaCs im Kolon unterbricht.
Zuletzt wurde die funktionelle Bedeutung der SGK3 in der Regulation des intestinalen Transports untersucht. Zuvor hatten Experimente im Xenopus-Expressionssystem gezeigt, dass die SGK3 eine Vielzahl von Transportern beeinflussen kann, u.a. den Glucosetransporters SGLT1. In SGK3-defizienten Mäusen (sgk3-/-) zeigte sich verglichen mit Wildtyp-Mäusen eine höhere Futteraufnahme und ein höheres Stuhltrockengewicht. Die Glukose-induzierten Ströme (Iglc) waren im Jejunum signifikant geringer in sgk3-/- - Mäusen als in Wildtyp-Tieren. Der Nüchtern-Blutzucker war in sgk3-/- -Mäusen signifikant niedriger. Der intestinale elektrogene Transport von Phenylalanin, Cystein, Glutamin und Prolin ware hingegen zwischen sgk3-/- und Wildtyp-Mäusen nicht verschieden. Daher kann gefolgert warden, dass die SGK3 für die intestinale Na+-gekoppelte Glukoseaufnahme erforderlich ist und dass eine verminderte Glukoseaufnahme für die Wachstumsretardierung und die niedrigen Blutzuckerwerte verantwortlich sein könnte, was zu einer kompensatorischen Zunahme der Futteraufnahme führen könnte
Glucocorticoids enhance intestinal glucose uptake via the dimerized glucocorticoid receptor in enterocytes
No full textGlucocorticoid (GC) treatment of inflammatory disorders, such as inflammatory bowel disease, causes deranged metabolism, in part by enhanced intestinal resorption of glucose. However, the underlying molecular mechanism is poorly understood. Hence, we investigated transcriptional control of genes reported to be involved in glucose uptake in the small intestine after GC treatment and determined effects of GC on electrogenic glucose transport from transepithelial currents. GR(villinCre) mice lacking the GC receptor (GR) in enterocytes served to identify the target cell of GC treatment and the requirement of the GR itself; GR(dim) mice impaired in dimerization and DNA binding of the GR were used to determine the underlying molecular mechanism. Our findings revealed that oral administration of dexamethasone to wild-type mice for 3 d increased mRNA expression of serum- and GC-inducible kinase 1, sodium-coupled glucose transporter 1, and Na(+)/H(+) exchanger 3, as well as electrogenic glucose transport in the small intestine. In contrast, GR(villinCre) mice did not respond to GC treatment, neither with regard to gene activation nor to glucose transport. GR(dim) mice were also refractory to GC, because dexamethasone treatment failed to increase both, gene expression and electrogenic glucose transport. In addition, the rise in blood glucose levels normally observed after GC administration was attenuated in both mutant mouse strains. We conclude that enhanced glucose transport in vivo primarily depends on gene regulation by the dimerized GR in enterocytes, and that this mechanism contributes to GC-induced hyperglycemia
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
SGK1-dependent upregulation of connective tissue growth factor by angiotensin II
No full textAngiotensin II has previously been shown to trigger fibrosis, an effect involving connective tissue growth factor (CTGF). The signaling pathways linking angiotensin II to CTGF formation are, however, incompletely understood. A gene highly expressed in fibrosing tissue is the serum-and glucocorticoid-inducible kinase SGK1. The present study explored whether SGK1 is transcriptionally regulated by angiotensin II and participates in the angiotensin II-dependent regulation of CTGF expression. To this end, experiments have been performed in human kidney fibroblasts and mouse lung fibroblasts from gene-targeted mice lacking SGK1 (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)). In human renal fibroblasts, SGK1 and CTGF protein expression were enhanced by angiotensin II (10 n M) within 4 h. In sgk1(+/+) mouse fibroblasts, SGK1 transcript levels were significantly increased after 4 h of angiotensin II treatment. Angiotensin II stimulated both transcript and protein abundance of CTGF in fibroblasts from sgk1(+/+) mice, effects significantly blunted in fibroblasts of sgk1(-/-) mice. In conclusion, angiotensin II stimulates the expression of SGK1, which is in turn required for the stimulating effect of angiotensin II on the expression of CTGF. Thus, SGK1 presumably contributes to the profibrotic effect of angiotensin II. Copyright (C) 2008 S. Karger AG, Basel
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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