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The high affinity sodium/glucose cotransporter (SGLT1) couples transport of Na+ and glucose. Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the first project, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Charged and polar residues and glucose/galactose malabsorption (GGM) missense mutations in TM IV were replaced with cysteine. Mutants exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. The results from mutants T156C and K157C suggest that TM IV participates in sugar interaction with SGLT1. This work has been published in Am J Physiol Cell Physiol 295 (1), C64-72, 2008. The crystal structure of Vibrio parahaemolyticus SGLT (vSGLT) was recently published (1) and showed discrepancy with the predicted topology of mammalian SGLT1 in the region surrounding transmembrane segments IV-V. Therefore, in the second project, we investigated the topology in this region, thirty-eight residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were individually replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. Based on the results from biotinylation of mutants in intact COS-7 cells, MTSES accessibility of cysteine mutants expressed in COS-7 cells, effect of substrate on the accessibility of mutant T156C in TM IV expressed in COS-7 cells, and characterization of cysteine mutants in TM V expressed in Xenopus laevis oocytes, we suggest that the region including residues 143-180 forms part of the Na+- and sugar substrate-binding cavity. Our results also suggest that TM IV of mammalian SGLT1 extends from residue 143-171 and support the crystal structure of vSGLT. This work has been published in Biochem Biophys Res Commun 378 (1), 133-138, 2009 Previous studies established that mutant Q457C human SGLT1 retains full activity, and sugar translocation is abolished in mutant Q457R or in mutant Q457C following reaction with methanethiosulfonate derivatives, but Na+ and sugar binding remain intact. Therefore, in the third project, we explored the mechanism by which modulation of Q457 abolishes transport, Q457C and Q457R of rabbit SGLT1 expressed in Xenopus laevis oocytes were studied using chemical modification, the two-electrode voltage-clamp technique and computer model simulations. Our results suggest that glutamine 457, in addition to being involved in sugar binding, is a residue that is sensitive to conformational changes of the carrier. This work has been published in Biophysical Journal 96 (2), 748-760, 2009. Taken together our study along with previous biochemical characterization of SGLT1 and crystal structure of vSGLT, we propose a limited structural model that attempts to bring together the functions of substrate binding (Na+ and sugar), coupling, and translocation. We propose that both Na+ and sugar enter a hydrophilic cavity formed by multiple transmembrane helices from both N-terminal half of SGLT1 and C-terminal half of SGLT1, analogous to all of the known crystal structures of ion-coupled transporters (the Na+/leucine transporter, Na+/aspartate transporter and lactose permease). The functionally important residues in SGLT1 (T156 and K157 in TM 4, D454 and Q457 in TM 11) are close to sugar binding sites.

Diabetes is a world-wide health problem with 415 millions of people suffering from the disease. Most diabetics are suffering from Type 2 Diabetes, which is preceded by insulin resistance in glucose utilizing tissues, such as adipose, liver, and muscle tissues. Diabetes is diagnosed when the insulin control of the glucose levels fails, which leads to high glucose levels in the blood. To better understand the insulin control of blood glucose, mathematical modeling has been used for many years to simulate the dynamics of glucose and insulin levels in the blood. Models have also been used to understand the intracellular insulin-signaling network in the insulin responding tissues. There have also been attempts to connect models from these different layers of control into a multi-level and multi-scale simulation model. However, to do such connections, several assumptions must be made about the comparability of the data from the different levels. Here, I aim for a deeper understanding of these assumptions and to use more advanced data for glucose uptake dynamics than in earlier work. I used data from the literature for the dynamics of glucose uptake in adipose and muscle tissues and improve the model in several steps to have a better agreement with these data. In particular, I refined the sub-division of the glucose uptake between the organs, to also account for liver uptake, a correction that implied a reduction by 50% for the muscle and adipose tissue glucose uptake. Unlike previous models, the updated model also describes blood flow. Finally, because of the connection to the intracellular level, the model can be used to simulate the response to anti-diabetic drugs.

The presented diploma work deals with the integration of children with diabetes in the company with a focus on education. The main objective of this work is to highlight the problems and situations with which it must contend daily pediatric patients with type I diabetes. The project should teach classmates disabled children, how his friends help some complications of their disease. The theoretical part of the work is the basis for the project itself and the practical part deals with both the project itself.

The presented diploma work deals with the integration of children with diabetes in the company with a focus on education. The main objective of this work is to highlight the problems and situations with which it must contend daily pediatric patients with type I diabetes. The project should teach classmates disabled children, how his friends help some complications of their disease. The theoretical part of the work is the basis for the project itself and the practical part deals with both the project itself and by questionnaire, the results pointed to the advisability of this project into the classroom.

La production endogène de glucose est une fonction cruciale au maintien de l’homéostasie glucidique dont les 2 dernières étapes sont la production de glucose par la glucose-6-phosphatase (G6Pase) et la sortie du glucose hors de la cellule par le transporteur facilité GLUT2. Les mécanismes dépendants de mouvements membranaires régulant ces deux étapes ont été étudiés. La régulation de la G6Pase par l’AMPc dépend de mouvements membranaires. Cependant les mécanismes moléculaires de cette régulation restaient à caractériser. Nous avons étudié l’hypothèse d’une phosphorylation directe des sous-unités de la G6Pase par la PKA. La PKA est capable d’induire l’activité G6Pase. Cependant, aucune phosphorylation des sous-unités G6Pase n’a pu être mise en évidence par phosphorylation in vitro, mutations dirigées de sites potentiels de phosphorylation ou analyse par spectrométrie de masse. En absence de Glut2, le glucose produit de novo sort des hépatocytes par une voie dépendante de mouvements membranaires, dont le mécanisme moléculaire n’est pas caractérisé. Cette voie vésiculaire n’est pas impliquée dans la sortie du glucose glycogénolytique. À l’inverse, 50% du glucose néoglucogénique sort des hépatocytes par une voie vésiculaire, probablement dépendante de la cavéoline-1. Par microscopie confocale à fluorescence, nous avons montré que la G6Pase se déplace dans la cellule vers la membrane plasmique et co-localise avec une partie de la cavéoline1 cellulaire. Les vésicules composées de cavéoline-1 et contenant la G6Pase pourrait donc constituer un lien entre le réticulum endoplasmique, lieu de production du glucose et la membrane plasmique, lieu de libération du glucose / Endogenous glucose production is a crucial function to maintain glucose homeostasis whose last two steps are glucose production by glucose-6-phosphatase (G6Pase) and glucose output by GLUT2. Regulations of both steps depend on membrane movements. In this work, we characterized the mechanisms of these regulations. Regulation of G6Pase by cAMP depends on membrane movements; however the molecular mechanisms of this regulation still have to be characterized. We hypothesized that PKA directly phosphorylated G6Pase subunits. We showed that PKA was able to enhance G6Pase activity. However, no phosphorylation of G6Pase subunits was evidenced by in vitro phosphorylation, directed mutagenesis of potentiel phosphorylation sites or mass spectrometry. In the absence of Glut2, the gluconeogenic glucose produced by hepatocytes is released through a pathway depending on membrane movements, which has not been characterised yet. This vesicular pathway was not involved in the output of glycogenolytic glucose. However, half of gluconeogenic glucose was released through a vesicular pathway, probably depending on caveolin-1. By confocal microscopy, we showed that G6Pase moved in cells and co-localized in part with cellular caveolin-1. Caveolin-1 vesicles containing G6Pase could thus constitute a link between the endoplasmic reticulum, site of glucose production, and the plasma membrane, site of glucose output

To prevent damaging ATP depletion during periods of intense activity‚ intrinsic mechanisms within skeletal muscle are activated and lead to myoprotection; a process known as muscle fatigue. It has been proposed that the primary mechanism of fatigue is a submaximal sarcoplasmic reticulum Ca2+ release and decreased force generation‚ however‚ what triggers this mechanism remains controversial. It is possible that glycogen may act as a trigger as studies have repeatedly shown a direct correlation between glycogen content at the beginning of activity and time to fatigue. In previous studies‚ a fatigue bout and/or period of fasting to deplete glycogen was used. However, this leaves investigators to differentiate between the effects of glycogen depletion methodology causing a metabolic stress and effects of glycogen itself. One objective of this M.Sc. project was to produce a low glycogen model without a prior metabolic stress that could forgo these limitations. It was extended to differentiate between the role of glycogen and extracellular glucose during fatigue as well as fatigue pre-conditioning (FPC)‚ a phenomenon in which fatigue resistance increases for about 2 hours after a first fatigue bout. During a single‚ first fatigue bout (one contraction every sec for 3 min) a mean decrease in glycogen from 95 to 20 μmol/g dry wt. had no effect on the decrease in tetanic [Ca2+]i‚ i.e. the [Ca2+]i during tetanic contractions‚ whereas removing glucose from the physiological solution led to a 46% greater decrease in tetanic [Ca2+]i than when glucose was present. During a subsequent fatigue bout (i.e. FPC) a greater amount of glycogen was used as glycogen content was 27% greater than prior to the first fatigue bout. When glycogen and/or glucose was limited‚ FPC was abolished. It is concluded that extracellular glucose is critical to prevent fatigue. Additionally, whereas glycogen is important for FPC‚ it appears to be much less important during a first fatigue bout initiated in absence of any prior metabolic stress.

RS1 ist ein 67-68 kD großes, ubiquitär exprimiertes Protein, das sich an der Innenseite der Plasmamembran befindet und in den Zellkern wandern kann. Durch immunhistochemischen Untersuchungen an Dünndarmschnitten der Maus konnte RS1 das erste Mal in dieser Arbeit im Kern und an der Membran von Enterozyten gezeigt werden. RS1 wird von einem intronlosen Single Copy Gen kodiert und ist fähig Ubiquitin über eine Ubiquitin-assoziierte (UBA) Domäne zu binden. Es reduziert die Konzentration einiger Proteine in der Plasmamembran. Durch Expressionsversuche in Xenopus Oozyten wurde gezeigt, dass RS1 die Menge des Na+-D-Glukosekotransporters SGLT1 in der Plasmamembran transkriptionsunabhängig reduziert. Entsprechend seiner dualen Lokalisation beteiligt sich RS1 aber auch an der Transkriptionsregulation im Zellkern. In der vorliegenden Arbeit konnten Informationen über die physiologische Funktion des membranassoziierten Regulatorproteins RS1 gewonnen werden. Nach Erstellung einer RS1-knock-out Maus wurde sichergestellt, dass ein erfolgreiches Rekombinationsereignis stattgefunden hatte und RS1 tatsächlich nicht mehr exprimiert wurde. Die RS1-knock-out Mäuse waren postnatal lebensfähig, vermehrten sich gut und entwickelten eine Fettsucht mit 30 % mehr Körpergewicht, 80 % mehr Fett und um 40 % vergrößerten Fettzellen. Bei den transgenen Mäusen war weder die Nahrungsaufnahme gesteigert, noch die motorische Aktivität verringert. In der Bürstensaummembran des Dünndarmepithels konnte bei den RS1-knock-out Mäusen die siebenfache Menge an Protein des Na+-abhängigen D-Glukosekotransporters SGLT1 detektiert werden, während die Konzentration des passiven Glukosetransporters GLUT2 in der basolateralen Membran nicht verändert war. Die Zunahme der SGLT1-Proteinmenge war posttranskriptional bedingt. Bei der RS1-knock-out Maus wirkt sich der in Oozyten beobachtete Effekt an der Plasmamembran aus, während der an konfluenten LLCPK1 Zellen gezeigte Effekt im Zellkern nicht zum Tragen kommt. Die transgenen Tiere resorbierten die doppelte Menge an D-Glukose im Dünndarm. Das spricht dafür, dass bei der RS1-knock-out Maus der „turnover“ des SGLT1 beeinflusst sein muss, da die siebenfache SGLT1-Proteinmenge einem verdoppelten Transport über den SGLT1 gegenübersteht. Die RS1-knock-out Mäuse zeigten normale Insulinspiegel und reguläre oralen Glukosebelastungstests. Bei gefütterten Mäusen waren die Serumleptinspiegel ähnlich wie bei Wildtypmäusen, die typische Reduzierung des Serumleptinspiegel konnte bei den Mäusen ohne RS1 aber nicht beobachtet werden. Untersuchungen an Fettzellexplantaten ergaben, dass die Sekretion von Leptin bei RS1- knock-out-Explantaten erhöht war, während die Leptinsynthese und die insulinabhängige Regulation der Leptinsekretion nicht verändert waren. Mit der RS1-knock-out Maus wurde ein neues Fettsuchtmodell geschaffen. RS1 spielt eine physiologisch wichtige Rolle bei der Regulation der D-Glukoseaufnahme im Darm. Der visceralen Adipositas liegt wahrscheinlich eine gesteigerte Nahrungsutilisation durch die verbesserte Glukoseaufnahme über den SGLT1 im Darm zugrunde. Die gesteigerte Glukoseabsorption ist ursächlich für den Anstieg der Fettmasse. Die Fettzellen vergrößern sich und sezernieren dann mehr Leptin. Es ist davon auszugehen, dass die RS1-knock-out Mäuse eine veränderte Nahrungsutilisation aufgrund der verbesserten Glukoseaufnahme im Dünndarm aufweisen. Die Adipositas demzufolge ein sekundärer Effekt. Gleichzeitig kann aber nicht ausgeschlossen werden, dass RS1 direkt auf die Zellen des weißen Fettgewebes wirkt und bei Wildtypmäusen die Sekretion des Leptins aus Vesikeln hemmt. / RS1 is a 67-68-kDa ubiquitously expressed protein, which is localized below the plasma membrane and within the nucleus. In this work, by immunohistochemistry on small intestine of wildtype mice, RS1 protein could be located to nucleus and plasma membrane of enterocytes the first time. An intronless single copy gene encodes RS1. RS1 is able to bind ubiquitin with an ubiquitin associated (UBA) domain, reduces concentration of some proteins in the plasma membrane. Coexpression experiments with RS1 and SGLT1 demonstrated that RS1 decreased the plasma membrane concentration of SGLT1 in Xenopus laevis oocytes. These effects were independent of transcription. In addition, consistent with the dual localization of RS1, transcriptional regulation has been observed in the nucleus. This work tries to elucidate the biological role of the plasma membrane–associated regulatory protein RS1. We generated RS1 knock-out mice and exploited them for experimental approaches. After generating RS1-knock-out mice, we verified correct homologous recombination and lack of expression of RS1 gene product. RS1-knock-out mice are viable, breed well, and are obese. Their body weight is increased by 30 %, body fat by 70 %, and mean fat cell volume by 40 % compared with wildtype animals. However, neither food intake was increased nor the motor activity was reduced in RS1-knock-out mice. In brush-border membranes of small intestine, the amount of protein of the Na+-dependent D-glucose transporter SGLT1 was increased sevenfold whereas protein expression of the glucose transporter GLUT2 did not differ. The increased amount of SGLT1 protein was not associated with higher expression of mRNA levels, indicating that regulation occurs on posttranscriptional level. At variance with the transcriptional upregulation of SGLT1 observed in confluent LLC-PK1 cells with reduced RS1 expression, the small intestinal upregulation of SGLT1 in RS1-knock-out mice is an effect at the plasma membrane, observed after expression in oocytes. The capacity of small intestinal D-glucose uptake in RS1-knock-out mice was 2fold increased compared to wildtype. This was due to a 7fold posttranscriptional upregulation of SGLT1 protein. The data suggest that the turnover of SGLT1 is changed. Plasma insulin levels were normal in RS1-knock-out mice. Plasma levels of glucose responded adequately upon oral glucose loading. In fed mice lacking RS1, serum leptin was similar as in wildtype, however, the typical starvation-induced reduction of serum leptin was not observed. In fat tissue explants of RS1-knock-out mice, leptin secretion was increased whereas expression of leptin as well as insulindependent regulation of leptin secretion were not changed. The RS1-knock-out mouse represents a novel model of obesity. RS1 plays a physiologically important role of regulation of D-glucose absorption in small intestine. Enhanced food utilization as a consequence of increased glucose absorption in the small intestine accounts for visceral type of adipositas of RS1-knock-out mice, probably. The increased glucose absorption leads to the rise in fat mass. The fat cells become larger and secret more leptin.