Glucose transporter GLUT4 allows glucose uptake into muscle and adipose cells. Insulin promotes recruitment and plasma membrane insertion of GLUT4 vesicles that can recycle constitutively. Obesity and type 2 diabetes mellitus are associated with defects in insulin-induced GLUT4 recruitment. Knowledge of alternative modes and steps of GLUT4 traffic in L6-GLUT4myc muscle cells may reveal possible targets for therapeutic intervention in insulin-resistant patients. Hypertonicity and Platelet Derived Growth Factor also increase surface GLUT4 levels but it was unknown if they tap on the same intracellular GLUT4 depots as insulin. We explored whether GLUT4 vesicles recycle using different compartments and mechanisms for the surface gain elicited by each stimulus. We hypothesized that all vesicle fusion steps require NSF but depend on individual v-SNAREs. Specifically, we tested effects of ATPase-deficient NSF or VAMP7 siRNA transfections, and endosomal ablation on GLUT4 traffic. We show that VAMP7 was required for basal and hypertonic recycling, while VAMP2 is exclusively used in response to insulin. As insulin action bifurcates downstream of phosphatidylinositol 3-kinase, we also hypothesized that the Rac-to-actin and Akt-to-AS160 branches regulate distinct GLUT4 traffic steps. For this we determined GLUT4myc localization in rounded myoblasts relative to a surface marker. Interfering with Rac, actin dynamics or actin-binding α-actinin4 maintained GLUT4 in a perinuclear region even under insulin-stimulation. Interfering with AS160 allowed significant GLUT4 accumulation beneath the membrane, but not fusion. We propose that actin dynamics and α-actinin4 are required for cortical GLUT4 tethering mechanisms, and AS160 contributes to GLUT4 docking/fusion. We confirmed that VAMP2 facilitates GLUT4 fusion, as tetanus toxin-based cleavage did not inhibit peripheral GLUT4 recruitment. Finally, AS160 targets Rab8A and Rab14 in muscle respectively affected GLUT4 availability for membrane fusion, and basal GLUT4 retention. This work will lead to future testing of strategies to selectively enhance vesicle availability, tethering, or surface fusion, for bypassing insulin resistance.

The original publication is available at www.springerlink.com.

Syftet med denna studie är att belysa vilka faktorer som ligger bakom företags beslut att själva äga eller outsourca hela eller delar av transportfunktionen. På en global marknad inser fler och fler företag att de inte besitter den kompetens som krävs i den allt tuffare konkurrensen. Det blir nödvändigt att samarbeta med andra företag för att kunna utnyttja deras kompetens och därigenom skapa konkurrensfördelar. Det har länge pågått en diskussion kring outsourcing som ett svar på detta ökade konkurrenstryck. Debatten kring in- och outsourcing har historiskt fokuserats mycket på kärnkompetens som en drivande faktor när företag ska besluta vad de själva ska producera kontra att lägga ut på marknaden. Röster har dock höjts för att utvidga förklaringsmodellen till att inkludera transaktionskostnadsperspektivet. Studien inriktar sig på den svenska livsmedelsbranschen och på hur företag har valt att organisera sin transportfunktion med avsikt på in- och outsourcing. Studien är genomförd med en kvalitativ ansats. Empiri samlades in genom tio semistrukturerade intervjuer från tio företag som alla är verksamma inom den svenska livsmedelsbranschen. Fyra företag är verksamma inom dagligvaruhandeln, tre företag är verksamma inom restaurant- och grossisthandeln och tre företag är producenter och leverantörer av dryckes- och färskvaror till både dagligvaru- och grossisthandeln. Bland företagen finns några av de största aktörerna på den svenska marknaden. En genomgång av tidigare forskning genomfördes för att konstruera en teoretisk referensram som sedan låg till grund för analysen. De begrepp som används i teoriavsnittet är Marknadsfaktorer, Specifika tillgångar, Osäkerhet/Mätning samt det Resursbaserade synsättet. Studien visar att företag in- eller outsourcar på grund av olika faktorer. Den visar i likhet med tidigare forskning att transaktionskostnadsperspektivet kombinerat med kärnkompetens ger en grund utifrån vilken beslut om in- och outsourcing kan förklaras. Vidare visar studien tydligare än tidigare studier att Marknadsfaktorer såsom svängningar i efterfrågan, fyllnadsgrad och transportavstånd spelar roll för företags sourcingbeslut. Detsamma gäller transaktionens Osäkerhet/Mätning, en hög grad av osäkerhet i transaktionen ökar företags benägenhet att insourca transportfunktionen.

The control of extracellular adenosine is crucial to the regulation of synaptic transmission and neuroprotection. Equilibrative nucleoside transporters (ENTs) are highly expressed in the hippocampus and widely accepted as critical regulators of adenosine tone. However, the mechanisms regulating the surface distribution and transport function of ENTs are largely unknown. Since ENT1 and ENT2 contain consensus sequences for phosphorylation by protein kinase CK2, and because this protein has been reported to regulate synaptic plasticity and ENT function in non-neuronal systems, the present thesis outlines the hypothesis that CK2-induced phosphorylation of ENTs is important for their cellular localization and thus the regulation of adenosine tone and synaptic transmission. Here, a functional interaction between adenosine CK2, ENTs and AMPA receptors in the hippocampus is reported. Western blot analysis shows that a variety of CK2 inhibitors (DMAT, TBB and DRB) significantly reduced the density of ENT1 and ENT2 proteins in hippocampal membrane fractions, suggesting that CK2-mediated phosphorylation of ENTs promotes their surface localization. In contrast, it was found that the ENT1 inhibitor NBTI significantly increased in the membrane localization of ENT1, relative to the control. Moreover, ENTs were found to immunoprecipitate with GluR1 and GluR2-containing AMPA receptors; and CK2 inhibitors caused a decrease in the membrane localization of GluR2 and GluR1 AMPA receptors. These results suggest a novel signaling complex linking CK2-regulated adenosine transport to AMPA receptor trafficking in the rat hippocampus. Although the physiological significance of these findings requires further investigation, this thesis provides insight into an adenosine regulation pathway that may be important for the regulation of synaptic transmission and neuroprotection in the rat hippocampus.

<p>Selective therapeutic targeting of tumors requires identification of differences between the homeostatic requirements of cancer and host cells. One such difference is the manner in which cancer cells acquire energy. Cancer cells often grow in an environment of local hypoxia; under these conditions tumor cells depend on glycolysis for energy, but are unable to perform oxidative phosphorylation. Many tumor cells, despite normoxic conditions, continue to perform glycolysis without oxidative phosphorylation. The net result of glycolysis without oxidative phosphorylation is twofold: the need to consume a greater amount of glucose than a non-cancerous host cell, and the burden of increased intracellular lactic acid. The proteins responsible for the transport of lactic acid in and out of cells are known as the monocarboxylate transporters (MCTs). Monocarboxylate Transporter 1 (MCT1) and Monocarboxylate Transporter 4 (MCT4) are the MCTs that play a major role in the transport of lactic acid. Tumor cells depend on MCT1 and MCT4 activity to excrete excess intracellular lactic acid to maintain neutral intracellular pH and homeostasis. Using human neuroblastoma and prostate cancer cell lines this work demonstrates that tumor cells can be selectively targeted tumor under conditions of hypoxia or acidosis in vitro with the drug lonidamine, with a small molecule inhibitor selective for MCT1, or with RNA interference of MCT1. Inhibition of MCT1 activity in neuroblastoma cells under acidic extracellular conditions results in intracellular acidification and cell death. MCT1 mRNA is expressed in human neuroblastoma and positively correlated with clinical risk profile. Inhibition of MCT1 activity in hypoxic prostate cancer cells results in a reduction of lactate excretion, decreased intracellular pH, inhibition of ATP production, and subsequent cell death. MCT1 expression in sections of human prostate tumors has been demonstrated to validate MCT1 as a target in prostate cancer.</p> <p>Through the Pasteur and Warburg effects, tumors have an increased demand for glucose. Some cancers store glycogen, but the reasons for this are largely unknown. It is hypothesized that tumor glycogen is used to promote tumor survival during transient hypoxia or low glucose, and that the mechanisms by which glycogen is stored is a potential therapeutic target in cancer. Tumors from human cell lines (WiDr, PC3, FaDu) have been grown in nude mice, sectioned and stained to measure glycogen storage. Using consecutive frozen sections, levels of hypoxia, glucose, lactate, ATP, and CD31, an endothelial cell marker, have been determined. These sections have been employed to elucidate the "architecture" of tumor metabolism in terms of vessel distance. Additionally, PAS-stained EF5 labeled human tumor samples were used to obtain calibrated hypoxia measurements to correlate with PAS. These studies demonstrate a correlation between hypoxia and the formation of glycogen deposits in human tumors and nude mouse xenografts. In cell culture, formation of glycogen deposits after exposure to hypoxia has been demonstrated, in addition to expression of glycogen synthase in human cancer cell lines.</p> <p>The development of novel selective cancer chemotherapeutics will require the identification of differences between cancerous cells and normal host cells to exploit as targets. Several differences in metabolism, including the need to excrete excess lactic acid and store glycogen under hypoxic conditions, are such targets. Novel therapeutics exploiting these targets should be effective against cancer cells and minimally toxic to host cells.</p> / Dissertation

<p>The environmental work in the Swedish transportation business was not very prioritized during the period up until 1990. If any environmental work occurred at all, it often entailed only necessary measures to meet the demands of environmental law.</p><p>All this changed almost over night roundabout 1990. The environmental issue had become politically important during the 1980s and environmental demands concerning large industrial and combustion plants started to wear off on the transportation business. In addition to this, competition between road and railway transportation sharpened due to political decisions on railway investments. The management of the national Swedish railway company SJ realized that this was a golden opportunity to become offensive in marketing, and that environment could become a success factor for the railway. By establishing an environmental policy and an environmental program, SJ took an initiative that large competitors and business representatives quickly tried to respond to by making investments on their own and by starting debates. Hereby, a rather unique situation occurred in that the transportation companies were driven by customers and society to make environmental investments and also give the business an environmental profile. This situation was the starting point for my study, where the aim has been to study the driving forces and trategies behind the environmental investments made in the Swedish transportation business during the 1990s. I have examined this by interviewing key persons in five companies representing different means of transport, i.e. the railway company SJ, the road transportation company DHL (earlier ASG), the road transportation company Schenker (earlier BTL and even earlier Bilspedition), the air transportation company SAS and the sea transportation company Stena Line. Apart from this, I have also studied the investment process in the chosen companies, and if environmental investments are different from other investments.</p><p>The method I have used in my study has been semi structured, qualitative interviews. A semi structured interview in this case means that I have used a certain amount of predefined questions. The responses from the interviewed person served as basis for follow up questions and new questions during the interview. A qualitative interview often consists of simple and straight questions being asked and complex and comprehensive answers being returned.</p><p>The results from the interviews could be summarised as follows: All of the companies in my study claim that it was the pressure of external and internal interests that made the starting point for their environmental work – even if the start appeared on different occasions depending on each company’s situation respectively. Any particularly deep analyses of the economical effects of environmental investments were not made on a regular basis. It rather seemed as if investments and other environmental decisions were based on intuitive feelings. It is also obvious that the pressure from competitors was a strong reason for continuing most of the companies´ environmental work – irrespective of the fact that the company used their environmental investments to create an offensive environmental profile, or to avoid to be criticized for lack of environmental commitment. It is possible to draw a number of conclusions from this study, some of them are general to the group of studied companies, and others are more specific to some of the companies.</p><p>The first, and perhaps most important, conclusion is that that the driving forces, for instance the pressure from owners, customers, competitors or society as such, were primary, and that the companies’ strategies was developed on this basis. One example is the Swedish railway company SJ, where the company management’s analysis showed both political pressure and obtainable market positions if the company developed and executed an offensive environmental strategy. The other companies in my study showed similar patterns even if their market positions of course varied.</p><p>The second conclusion is that external profiling also leads to internal pressure within the company. This pressure may be consciously and directed by management via education, seminars etc, or occur on its own due to that the company’s environmental work leads to an interest and commitment amongst employees. The best examples of this are the companies SJ and ASG.</p><p>A third conclusion is that in many cases, the environmental investments could be compared to a one way road. Some decisions are irrevocable, at least if the company wants to maintain its credibility within the environmental area. For instance, it is equally impossible in practise for SJ to back on the decision to buy environmentally labelled electricity, as for DHL and Schenker to return to diesel fuel with high level of sulphur.</p><p>The last conclusion is that economical arguments for environmental investments offer a very strong explanation model to which investments were made and which were not. This conclusion seems to be generally valid even if differences in grade exist, depending on if the investments were made with the purpose to be offensive or defensive. No follow up analyses of the economic outcome of the investments have - with a few exceptions – however not been made.</p><p>Environmental investments consist of physical and image enhancing (i.e. soft) investments. This study shows that physical environmental investments and image enhancing investments have been approached in the same way, but it is harder to quantify the image enhancing investments in economical terms.</p> / <p>Miljöarbetet i transportnäringen var inte särskilt påtagligt under perioden fram till 1990. I den mån det överhuvud taget ägde rum något sådant arbete handlade det oftast om åtgärder som var nödvändiga för att klara lagstiftningens krav.</p><p>Detta ändrades nästan ”över en natt” runt 1990. Miljöfrågan hade blivit viktig rent politisk under 1980-talet och miljökrav som tidigare hade ställts gentemot stora industri- och förbränningsanläggningar hade börjat smitta av sig även på transportnäringen. Dessutom kom konkurrensen mellan landsvägstransporter och järnväg att skärpas genom politiska beslut om järnvägsinvesteringar. SJ:s ledning insåg att detta var ett gyllene tillfälle att gå på offensiven marknadsmässigt, samt att miljö var en framgångsfaktor för järnvägen. Genom antagandet av en miljöpolicy och ett miljöprogram tog man ett initiativ som stora konkurrenter och branschföreträdare som Bilspedition, Sjöfartsverket, Åkeriföreningen med flera snabbt försökte kontra, dels med egna investeringar, dels i debattform. Därmed hade den för Europa rätt unika situation uppstått att transportföretagen drevs av såväl kunder och samhälle som konkurrenter att investera i miljö och allt mer tydligt profilera verksamheten ur miljösynpunkt. Denna situation var utgångspunkten för min studie, där syftet har varit att klarlägga de drivkrafter och strategier som legat bakom de miljöinvesteringar som genomfördes i den svenska transportbranschen under 1990-talet. Jag har exemplifierat detta genom att intervjua nyckelpersoner i fem företag, representerande olika trafikslag, nämligen SJ, DHL (dåvarande ASG), Schenker (dåvarande Bilspedition respektive BTL), SAS och Stena Line. Utöver huvudsyftet har jag också undersökt hur investeringsprocessen ser ut i företagen, och om miljöinvesteringar skiljer sig från andra investeringar.</p><p>Metoden som använts i studien har varit semistrukturerad, kvalitativ intervjumetodik. Semistrukturerad intervju innebar i detta fall att jag har använt ett antal frågor nedskrivna på papper som utgångspunkt. Respondentens svar utgjorde sedan underlag för uppkomsten av följdfrågor och nya frågor under intervjuns gång. Kvalitativ intervju utmärks ofta av att enkla raka frågor ställs och komplexa, innehållsrika svar kommer tillbaka.</p><p>Resultatet av intervjuerna kan sammanfattas enligt följande: Samtliga studerade företag anger att det var intressenttrycket som var utgångspunkten för miljöarbetet – även om starten av det skedde vid olika tillfällen, beroende på respektive företags situation. Några ärskilt djupgående analyser av miljöinvesteringarnas företagsekonomiska effekter gjordes inte mer än undantagsvis, det förefaller snarare som man gick efter något slags intuitiv känsla av vad som var nödvändigt. Det är också uppenbart att konkurrenttrycket var en starkt bidragande orsak till det fortsatta miljöarbetet inom de flesta av företagen – oavsett om man använde miljöinvesteringarna för att skapa sig en offensiv miljöprofil, eller för att undvika alltför hård kritik för bristande miljöengagemang. Utifrån resultaten i denna studie kan man dra ett antal slutsatser, flera av dem generella för samtliga studerade företag, några andra mer specifika för vissa av dem.</p><p>Först, och kanske viktigast, kan man konstatera att drivkrafterna, t.ex. trycket från ägare, kunder, konkurrenter eller samhället i övrigt, var de primära och strategin utformades utifrån detta. Exempel på detta är SJ, där företagsledningens analys visade att det fanns både ett politiskt tryck samt marknadsmässigt uppnåeliga positioner om</p><p>man utvecklade och genomförde en offensiv miljöstrategi. De övriga intervjuade företagen uppvisade likartade mönster, även om positionerna på marknaden givetvis varierade.</p><p>Den andra slutsatsen är att en extern profilering också leder till ett internt tryck inom företaget. Detta tryck kan vara medvetet, och dirigerat från företagsledningen via utbildning, seminarier etc eller uppstå av sig självt genom att miljöåtgärderna skapar ett intresse och engagemang hos företagets anställda. De bästa exemplen på detta är SJ och ASG.</p><p>En tredje slutsats är att miljöinvesteringarna i många fall kan liknas vid en enkelriktad väg. En del beslut är oåterkalleliga, i alla fall om företaget ska bibehålla sin trovärdighet på miljöområdet. Det är t.ex. i praktiken omöjligt för SJ att backa från beslutet att köpa miljömärkt elkraft, liksom för DHL och Schenker att gå tillbaka till dieselbränsle med hög svavelhalt.</p><p>Den sista slutsatsen är att de ekonomiska argumenten för miljöinvesteringar erbjuder en mycket stark förklaringsmodell till vilka investeringar som genomfördes respektive inte genomfördes. Denna slutsats förefaller vara generellt användbar, även om valörskillnader självklart finns, beroende på om investeringarna genomfördes i offensivt eller defensivt syfte i de olika företagen. Några efteranalyser av det ekonomiska utfallet har dock – med ytterst få undantag – inte gjorts.</p><p>Miljöinvesteringar består av såväl fysiska som imagestärkande (i.e. mjuka) investeringar. I studien har det framkommit att fysiska miljöinvesteringar och imagestärkande miljöinvesteringar i stort sett hanterats likadant, dock är det svårare att kvantifiera de imagestärkande investeringarna rent ekonomiskt.</p> / Report code: Liu-Tek-Lic-2007: 49.

The glucose transporter 4 (GLUT4) is the major insulin-responsive glucose transporter in adipose and muscle tissues. Although the early steps in the insulin signaling pathway governing translocation of GLUT4 to the plasma membrane are well understood, the final steps in this pathway are not. TUG is a protein which has been shown to affect trafficking of GLUT4 both in the basal state and in response to insulin. One protein-protein interaction between TUG and the large cytosolic loop of GLUT4 has previously been identified. Based on reports of the requirement of the GLUT4 N-terminal domain for its proper targeting to the plasma membrane, we postulated that an interaction might also exist between TUG and the N-terminal domain of GLUT4, and we tested this hypothesis using two sets of pull-down experiments. In the first set, using the N-terminal domain of GLUT4 fused with glutathione S-transferase (GST), we were able to pull TUG down from the lysates of TUG-transfected HEK 293 cells. TUG was also pulled down by the GLUT4 cytosolic loop and, to a much lesser extent, its C-terminal domain. However, there was no specific interaction between these fusion proteins and the lysates of cells transfected with a truncated form of TUG lacking its own N-terminal domain. In the second set of experiments, using a biotinylated synthetic GLUT4 N-terminal peptide, we pulled down a protein detected by an anti-TUG antibody and running at ~64 kDa, a slightly higher molecular weight than wild-type TUG. We believe that this band represents modified full-length TUG. This interaction was not seen using synthetic GLUT4 N-terminal peptide mutated at 4 amino acids previously identified as necessary for proper GLUT4 retention and insulin-responsive trafficking. We conclude that TUG interacts not only with the large cytosolic loop of GLUT4, but also with the N-terminal domain of GLUT4, and that this latter interaction can be disrupted by mutations in GLUT4 that cause defective trafficking, suggesting that this interaction is critical for GLUT4 intracellular retention and insulin-responsive GLUT4 trafficking.

Στο κεντρικό νευρικό σύστημα (ΚΝΣ), η ντοπαμίνη ελέγχει σημαντικές φυσιολογικές λειτουργίες όπως η έκκριση ορμονών, η ρύθμιση της κίνησης, γνωστικές πορείες και περίπλοκες συμπεριφορές που έχουν σχέση με το συναίσθημα και την ανταμοιβή. Η επαναπρόσληψη της ντοπαμίνης διαμέσου του μεταφορέα της νευρωνικής πλασματικής μεμβράνης (DAT) είναι σημαντική για τη διατήρηση της ομοιόστασης της στο ΚΝΣ. Πιστεύεται ότι ο DAT ελέγχει την ένταση και τη διάρκεια της ντοπαμινεργικής νευροδιαβίβασης. Ο DAT αποτελεί μοριακό στόχο θεραπευτικών ουσιών για την θεραπεία νοητικών δυσλειτουργιών όπως η σχιζοφρένεια και η κατάθλιψη. Επιπροσθέτως είναι στόχος εθιστικών ουσιών όπως η κοκαΐνη και η αμφεταμίνη. Οι ψυχοδιεγερτικές και οι θεραπευτικές ουσίες δεσμεύονται στον DAT, αλλάζουν την λειτουργία του και ως εκ τούτου επιτείνουν την ένταση και διάρκεια της ντοπαμινεργικής νευροδιαβίβασης. Η παρεγκεφαλίδα δέχεται μια κατεχολαμινεργική νεύρωση η οποία θεωρείται ότι είναι νοραδρενεργική. Εν τούτοις βιοχημικές, φαρμακολογικές και ανατομικές μελέτες υποδεικνύουν ότι επίσης δέχεται μια μικρή ντοπαμινεργική νεύρωση από την κοιλιακή περιοχή της καλύπτρας και την συμπαγή μοίρα της μέλαινας ουσίας. Πρόσφατες ανοσοϊστοχημικές μελέτες μας για τον DAT υπέδειξαν ότι νευρώνες της παρεγκεφαλίδας μπορεί να εκφράζουν τον μεταφορέα. Στην παρούσα μελέτη ο εντοπισμός του μεταφορέα της ντοπαμίνης (DAT) στην παρεγκεφαλίδα φυσιολογικών μυών, τόσο σε επίπεδο πρωτεΐνης όσο και σε επίπεδο mRNA έγινε με την μέθοδο Western Blot, IP και RT-PCR αντίστοιχα. Η πρωτεΐνη ανιχνεύθηκε στο κλάσμα των συναπτοσωμάτων, ενώ το mRNA σε ολικό εκχύλισμα παρεγκεφαλίδας. Τα αποτελέσματα έρχονται να επιβεβαιώσουν την ύπαρξη ντοπαμινεργικών κυττάρων στην παρεγκεφαλίδα. / In the Central Nervous System (CNS) the neurotransmitter dopamine (DA) controls important functions including hormone secretion, locomotion, cognitive processes and complex behaviours that are associated with emotion and reward. Dopamine uptake through the neuronal plasma membrane DA transporter is essential for the maintenance of normal DA homeostasis in the brain. It is believed that DAT controls the intensity and the duration of dopamine neurotransmittion. DAT is the molecular target for therapeutic agents used in the treatment of mental disorders, such as schizophrenia and depression. In addition, DAT is the target for cocaine and amphetamine. Psychostimulants and therapeutic substances alter its transporter function and therefore prolong the intensity and duration of dopaminergic neurotransmittion.The cerebellum receives a catecholaminergic input that is generally accepted to be noradrenergic. However, biochemical, pharmacological and anatomical evidence indicate that the cerebellum also receives a small dopaminergic input from ventral tegmental area and substantia nigra pars compacta. Recent immunohistochemical studies have indicated that cerebellar neurons express the transporter. In the current study, cerebellar dopamine transporter protein and mRNA were studied using the methods of western blotting and RT-PCR. Our results confirm the existence of dopaminergic neurons in cerebellum.

Solute carriers (SLCs) comprise the largest group of transporters in humans and there are currently 52 SLC families. They are embedded in cellular membranes and transport numerous molecules; defects in many of the genes encoding SLCs have been connected to pathological conditions, and several SLCs are potential drug targets. The SLC38 family has in total eleven members in humans and they encode transporters called SNATs. In paper I and paper II, we reported molecular and functional characterization of Slc38a7 and Slc38a8, two of the previous orphan members in the family which we suggested to be named SNAT7 and SNAT8, respectively. Using in situ hybridization and immunohistochemistry, these transporters showed similar expression pattern and localized to neurons in the brain For functional characterization proteins were overexpressed in X. laevis oocytes and an Uptake Assay and electrophysiological recordings showed preferred transport of L-glutamine, L-histidine, L-alanine, L-asparagine, L-aspartate and L-arginine for SNAT7. A similar pattern was seen for SNAT8 in a slightly different order of affinities. We classified SNAT7 as a system N transporter and SNAT8 as belonging to system A, and suggests that SNAT7 and SNAT8 could play a role in the glutamine/glutamate(GABA) cycle (GGC) in the brain. Furthermore, we studied the vesicular B0AT3 (Slc6a17) transporter in paper III, and the sodium-coupled amino acid transporter B0AT2 (Slc6a15) in paper IV. Tissue expression studies showed similar localization of Slc6a17 and Slc6a15 mRNA using in situ hybridization and real-time PCR. In paper III, vesicular localization of B0AT2 was shown in both excitatory and inhibitory neurons. When challenging the monoaminergic system with drugs both Slc6a17 and Slc6a15 were upregulated. Suggested roles for the transporters are thereby in synaptic remodeling by regulating the availability of free amino acids used as precursors needed in neurotransmitter synthesis. Moreover, in paper IV, immunohistochemistry showed B0AT3 localization to neurons, astrocytes and epithelial cells of the choroid plexus. Leucine injections caused a smaller reduction of food intake as well as higher neuronal activation in the paraventricular hypothalamic nucleus in Slc6a15 KO mice, compared with wild type mice. This suggests B0AT2 involvement in the anorexigenic effects of leucine.

The enrichment of egg with folate is a viable option for supplying the general population of a food product rich in natural folates. However, attempts to increase the concentration of folate in egg beyond the achieved level of enrichment had been unsuccessful because egg folate reached a maximum plateau when folic acid (FA) was increased in the diet. Thus, experiments were conducted to determine the factors regulating the deposition of dietary FA into the eggs. In the first study, the effect of feeding equimolar intake of FA and 5-methyltetrahydrofolate (5-methylTHF), the biologically active form of folate; on egg folate concentrations, indices of folate status, and activities of folate-dependent enzymes was evaluated. Folic acid and 5-methylTHF demonstrated equivalent effects in enhancing the egg folate concentrations and improving the indices of folate status in the laying hen. The activities of folate-dependent enzymes were similar between the two forms of folate except for hepatic dihydrofolate reductase (DHFR) activity which increased in FA-fed birds compared to 5-methylTHF-fed birds. However, this demonstrated the ability of the laying hen to metabolically convert FA into its biologically active forms. Therefore, the influence of intestinal FA absorption in the regulation of FA deposition in the egg was subsequently evaluated. Using the in vitro everted intestinal sac technique, FA was absorbed in all regions of the intestine. Absorption was maximum at acidic pH 6.0, and increased in the duodenum and jejunum compared to the ileum and cecum. The rate of FA absorption in the jejunum diminished at higher FA concentrations. Therefore, further study was conducted to determine the regulation of FA absorption when levels of FA in the laying hen diet are increased. Supplementation of increased FA levels resulted to a down-regulation of FA absorption in the duodenum, but not in the jejunum of the laying hen. This down-regulation was not associated to a decreased mRNA gene expression of the duodenal folate transporters. Overall, decreased intestinal rate of FA absorption possibly associated to a post-transcriptional or translational regulation of specific folate transporters in the intestine of the laying hen may contribute to the saturation in the egg folate concentration.