7.5 Conclusions
In the present study, the Ussing chamber technique was adapted to describe electrogenic activities of isolated carp intestine, especially a phlorizin sensitive electrogenic cotransporter activity that can be only explained by SGLT1. This activity mainly took part in the mid intestine of carp, in particular in lower mid intestine. Partial sequences of carp SGLT1, claudin-1, -2, -3b, -3c, -7, -11, -23, and -30 were isolated, their tissue expression profiles were described, and their expression levels along the gut axis were observed on the transcription level. SGLT1 is a highly conserved gene and carp SGLT1 is highly similar to the gene of zebrafish. Compared to its expression in other tissues, the mRNA expression of carp SGLT1 was high in intestine and kidney. In gut it was higher especially in the upper mid intestine. Carp claudins showed a tissue-specific expression pattern and in the intestine most of the genes were highly expressed in posterior part of gut.
Intestinal levels of expression of carp SGLT1 and claudin genes were affected by challenges with pathogens and during a feeding experiment. In a systemic infection with KHV intestinal expression of carp SGLT1 mRNA was decreased and transcriptional levels of claudin-2, -3c, -11, and -23 were increased. Alterations in the expression of claudin genes were also observed after a feed modulation and a challenge of carp with Aeromonas hydrophila. A change from normal feed to experimental feeds increased mRNA levels of claudin-3c, -7, -23, and 30 in the posterior intestine and reduced the transcriptional level of claudin-2 in middle intestine. Supplementation of -glucan to the experimental diet did not influence mRNA expression of SGLT1 or claudin genes. However a fortification of the diet with this polysaccharide which is composed of D-glucose monomers and which was derived from Saccharomyces cerevicae cell walls was associated with differential responses of claudins to an administration of Aeromonas hydrophila into the gut. In fish fed without -glucan supplementation, an intubation of the bacterium elevated the levels of claudin-11 and -30 mRNA while in carp which received the -glucan enriched diet, the challenge with the bacterium raised mRNA levels of claudin-3c and -23. Additionally, feeding with -glucan seemed to protect carp from inflammation processes associated with the bacterial intubation.
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Summary
Hamdan Syakuri
Studies of Intestinal Barrier Functions of Common Carp, Cyprinus carpio, under Feeding Modulation and Pathogen Challenge
The epithelium which covers the mucosal surface of the intestine of animals has important barrier functions which include the selective uptake of nutrients, the traffic of ions and water, and the protection of intestinal tissues as well as tissue beneath the mucosa from luminal antigens, toxins, or pathogenic invaders. For an improvement of fish production and fish health in aquaculture, more comprehensive knowledge on the diverse functions of the intestine in fish is needed. In aquaculture, supplies with highly digestable, supportive feed on a commercial basis and the prevention/ treatment of outbreaks of infectious diseases are issues which mainly challenge the production of fish including common carp for along period. The present work therefore was aimed to investigate the transporter protein SGLT1 and inter membrane proteins from the claudin family as two important elements of the intestinal barrier which are involved in selective permeability and the protective function of the intestine.
The Ussing chamber technique was implemented to describe electrogenic activities across the isolated carp intestine, in particular SGLT1 activity. PCR-based molecular assays were used to identify carp SGLT1 and claudin genes, describe their transcriptional profiles, and evaluate the modulation of their expression during the challenge with pathogens or the change of feeding. The potential dietary immunomodulant -glucan and two important disease causaing agents in carp aquaculture, koi herpesvirus (KHV) and Aeromonas hydrophila were used as experimental models.
In carp intestinal glucose uptake that is mediated by SGLT1 exclusively takes place in the middle intestine, especially in the lower mid gut. Because it is known as a highly conserved gene, carp SGLT1 is very similar to the gene from other piscine
9 )
species, in particular from zebrafish. The mRNA of this gene was highly expressed in the intestine, in particular in the upper mid intestine, in kidney, and at lower levels it was expressed in other tissues such as brain, liver, and gills. Intestinal transcription of the carp SGLT1 gene was reduced under KHV infection and it was not altered during a challenge of carp gut with Aeromonas hydrophila and after the modulation of feeding. Partial DNA fragments of eight carp claudin genes were isolated. Some of the claudin genes of carp such as claudin-7 and -11 seemed to be expressed in various tissues while some others like claudin-2 and -23 showed more tissue-specific expression profiles. In carp intestine, an infection with koi herpesvirus (KHV) increased mRNA expression of some claudin encoding genes such as claudin2, 3, -11, and -23. A change of the experimental feeding increased transcriptional levels of claudin-3c, -7, -23, and 30 in the posterior intestine and reduced the expression of claudin-2 mRNA in the middle intestine. However feeding of carp with -glucan did not influence mRNA expression of the genes encoding tight junction proteins, it appeared to prevent an intestinal inflammation and to differentiate the transcriptional response of claudin genes due to a bacterial challenge. Shortly after intubation of the bacterium carp that received a diet without -glucan showed an upregulation of claudin-11 and -30 transcriptions while carp which were fed with a -glucan containing diet indicated an upregulation of claudin-3c and -23.
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Zusammenfassung
Hamdan Syakuri
Studien zur intestinalen Barriere von Karpfen (Cyprinus carpio) nach Futterumstellung und unter Infektion mit Pathogenen
Das Epithel, das die mukosale Oberfläche im Darm von Tieren auskleidet, erfüllt wichtige Barrierefunktionen, die die selektive Aufnahme von Nährstoffen und den Transport von Ionen und Wasser ebenso einschließt wie den Schutz der Mukosa und der Gewebe unter der Mukosa vor luminalen Antigenen, Toxinen oder vor pathogenen Eindringlingen. Zur Verbesserung von Fischproduktion und Fischgesundheit in der Aquakultur ist eine umfangreichere Kenntnis über diese unterschiedlichen Funktionen im Darm von Fischen notwendig. In der Aquakultur sind insbesondere die Versorgung der Fische mit hoch verdaulichem, gehaltvollem und preiswertem Futter, der Schutz der Fische vor Infektionserkrankungen bzw. die Behandlung von Infektionserkrankungen Herausforderungen, die die Produktion von Fischen einschließlich des Speisekarpfens seit langem erheblich belasten. Die vorliegende Arbeit untersuchte deshalb den Kotransporter SGLT1 und Intermembran-Proteine aus der Claudin-Familie als zwei wichtige Elemente der intestinalen Barriere, die an der selektiven Permeabilität sowie der Schutzfunktion des Darms beteiligt sind.
Untersuchungen in Ussingkammern wurden zur Beschreibung der elektrogenen Vorgänge an isoliertem Darmgewebe von Karpfen, insbesondere der Aktivität von SGLT1 etabliert. Außerdem wurden molekulargenetische, auf PCR basierende Untersuchungen ausgeführt, um das SGLT1 codierende Gen und Claudin codierende Gene zu identifizieren, das Transkriptionsmuster dieser Gene in Karpfengeweben zu beschreiben, sowie um die Modulation der Expression dieser Gene unter Infektion von Karpfen mit Pathogenen oder nach einer Futterumstellung zu untersuchen. Als Versuchsmodelle wurden die Verfütterung des Immunmodulators Beta-Glukan als Futterzusatz, sowie die beiden in der Aquakultur
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von Karpfen bedeutsamen Pathogene Koi Herpesvirus (KHV) und Aeromonas hydrophila verwendet.
Der durch SGLT1 vermittelte Glukosetransport erfolgt bei Karpfen im Mitteldarm, insbesondere im posterioren Abschnitt des Mitteldarms. Weil das SGLT1 codierende Gen als hoch konserviert bekannt ist, weist die Nucleotidsequenz des SGLT1 codierend Gens von Karpfen weist eine hohe Übereinstimmung mit der Nukleotidsequenz des Gens von anderen Fischarten und insbesondere vom Zebrafisch auf. Die mRNA dieses Gens war im Darm, und hier vor allem im vorderen Mitteldarm hoch exprimiert, ebenso wie in der Niere; in geringerem Maße war es in anderen Organen, wie Gehirn, Leber und Kiemen exprimiert. Die Expression dieses Gens war bei Karpfen unter Infektion mit KHV verringert, sie war aber nach einer oralen Verabreichung von Aeromonas hydrophila oder nach einer Futterumstellung nicht verändert. Von acht Claudin codierenden Genen wurden Genfragmente isoliert.
Einige dieser Claudin-Genen, wie Claudin-7 und -11 waren in unterschiedlichen Geweben von Karpfen exprimiert, während andere Gene, wie Claudin-2 oder -23 mehr gewebespezifische Expressionsprofile aufwiesen. Bei Karpfen Darm, hatte eine Infektion mit Herpesvirus koi (KHV) eine Erhöhung der mRNA Expression einiger Claudin kodierenden Gene wie Claudin-2, -3, -11, und -23. Eine Umstellung des im Experiment verabreichten Futters hatte eine Erhöhung der Transkriptonsraten von Claudin 3c, -7, -23 und -30 im Enddarm zur Folge und eine Verringerung der Expression von Claudin-2 mRNA im Mitteldarm. Das Verfüttern von Beta-Glukanen beeinflusste die mRNA –Expression der Gene, die für die untersuchten Transmembranproteine kodieren, jedoch nicht. Allerdings schien die Verfütterung von Beta-Glukanen bei Karpfen nach oraler Verabreichung von Aeromonas hydrophila das Auftreten von Entzündungsreaktionen zu verhindern und eine Expression von Claudin-Genen zu beeinflussen. Kurze Zeit nach der Verabreichung der Bakterien war im Darm von Karpfen, die mit einer Diät ohne Beta Glukanzusatz gefüttert wurden, eine erhöhte Transkription von Claudin-11 und -30 zu erkennen, während im Darm von Karpfen, die eine Diät mit Beta-Glukan Zusatz erhielten, Claudin 3c und -23 hochreguliert waren.
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