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Alcohol is a neuroactive molecule that is able to exert variable and often detrimental effects on the developing brain, resulting in a broad range of physiological, behavioural, and cognitive phenotypes that characterize ‘fetal alcohol spectrum disorders’ (FASD). Factors affecting the manifestation of these phenotypes include alcohol dosage, timing of exposure, and pattern of maternal alcohol consumption; however, the biological processes that are vulnerable to ethanol at any given neurodevelopmental stage are unclear, as is how their disruption results in the emergence of specific pathological phenotypes later in life. The research included in this thesis utilizes a C57BL/6J (B6) mouse model to examine the changes to gene expression and behaviour following a binge-like exposure to ethanol during synaptogenesis, a period of neurodevelopment characterized by the rapid formation and pruning of synaptic connectivity that correlates to brain development during the human third trimester. B6 pups were treated with a high dose (5 g/kg over 2 hours) of ethanol at postnatal day 4 (P4), P7, or on both days (P4+7). Mice were evaluated using a battery of behavioural tests designed to assess FASD-relevant phenotypes, and showed delayed achievement of neuromuscular coordination, hyperactivity, increased anxiety-related traits, and impaired spatial learning and memory. Gene expression analysis identified 315 transcripts that were altered acutely (4 hours) following ethanol exposure. Up-regulated transcripts were associated with cellular stress response, including both pro- and anti-apoptotic molecules, as well as maintenance of cell structural integrity. Down-regulated transcripts were associated with energetically costly processes such as ribosome biogenesis and cell cycle progression. Genes critical to synapse formation were also affected, as well as genes important for the appropriate development of the hypothalamic-pituitary-adrenal axis. Additionally, gene expression changes within the adult brain of mice treated with ...

Die AMP-aktivierte Proteinkinase (AMPK) ist ein Enzym, das den Energiehaushalt der Zelle reguliert: AMPK erhöht die intrazelluläre ATP Konzentration, indem es energieverbrauchende Prozesse bremst und energieproduzierende fördert. Im Falle erschöpfter Energiereserven starten Erythrozyten ihr Apoptose-Programm, auch Eryptose genannt. Initiiert durch eine Zunahme der zytosolischen Ca2+-Konzentration ([Ca2+]i) kommt es zu Zellschrumpfung und der Exposition von Phosphatidylserin (PS) auf der Außenseite der Erythrozytenmembran. Die vorliegende Studie untersuchte, welche Rolle die AMPK in der Regulation der Eryptosis spielt. Die Expression von AMPK in den Erythrocyten wurde mittels der Western Blot Methode und Konfokalmikroskopie nachgewiesen. Die intrazelluläre Ca2+-Konzentration ([Ca2+]i), das Zellvolumen der Erythrozyten sowie Phosphatidylserin auf der Außenseite der Membran wurden mittels Durchflusszytometrie (FACS) ermittelt. Glukosemangel im Extrazellulärmedium führte zu einer Erhöhung von [Ca2+]i, verkleinerte das Zellvolumen und erhöhte den Anteil von PS in der äußeren Membran. Versuchsreihen mit dem AMPK-Inhibitor Compound C (20 µM) ergaben, dass bei Anwesenheit von Glukose keine signifikante Veränderung der Eryptoserate eintritt. Hingegen verstärkte Compound C bei Glucoseentzug die Eryptose deutlich. Das Ca2+-Ionophor Ionomycin führt zu einer Erhöhung von [Ca2+]i, welche wiederum die Eryptose auslöst. Dieser Effekt wurde durch den AMPK-Aktivator 5-Aminoimidazol-4-carboxamid-1-beta-D-ribofuranosid (AICAR; 1 mM) abgeschwächt. Verglichen mit Erythrozyten des Wild-Typs (ampk+/+), waren Erythrozyten von AMPK&alpha1- defizienten Mäusen (ampk-/-) signifikant anfälliger für Energiemangel bedingte Eryptose. Die ampk-/--Mäuse hatten trotz exzessiver Retikulozytose eine Anämie und litten unter schwerer Splenomegalie, was auf einen erhöhten Erythrozytenumsatz hinweist. Diese Beobachtungen zeigen, dass AMPK die Lebensdauer von zirkulierenden Erythrozyten beeinflusst. AMP-aktivierte Protein Kinase, eine Serin/Threonin-Kinase, wird durch Energiemangel aktiviert. Sie stimuliert die Energieprodukion und hemmt den Energieverbrauch. Dieser Effekt der AMPK wurde insofern für die GLUT-Familie der erleichterten Glucosetransporter nachgewiesen, als die Aktivierung der AMPK zu einem erhöhten Glukoseeinstrom druch GLUT-Transporter führt. Die vorliegende Studie untersuchte die Möglichkeit, ob die AMPK eventuell den Na+ gekoppelten Glukosetransport durch SGLT1 (SLC5A1) reguliert. Dazu wurde die cRNA von SGLT1 in Xenopus laevis-Oozyten mit und ohne AMPK exprimiert. Der elektrogene Symport von Na+/Glucose wurde mithilfe der Zwei-Elektroden-Spannungsklemme (DEVC) gemessen. In SGLT1- exprimierenden Oozyten konnte durch die Zugabe von Glukose zum extrazellulären Bad ein Strom (Ig) generiert werden. Dieser erreichte seine halbmaximale Stärke bei einer Konzentration von (KM) = 650 µM Glucose. Bei Oozyten, in die lediglich Wasser oder die konstitutiv aktive gammaR70QAMPK (alpha1beta1gamma1(R70Q)) injiziert wurde, ließ sich dieser Strom durch Glukosezugabe nicht erzeugen. Die Koexpression von gammaR70QAMPK hatte keinen Einfluss auf KM, erhöhte aber signifikant den maximalen Strom Ig max (= 1.7 fach). Die Koexpression des AMPK-Wildtyps oder der inaktivierten alphaK45RAMPK Mutante (alpha1(K45R)beta1gamma1) wirkte sich auf Ig nicht messbar aus. AICAR (1 mM), Phenformin (1 mM) und A-769662 (10 µM) erhöhten die SGLT1-Expression in der Zellmembran von Caco-2 Zellen, wie durch Konfokalmikroskopie und Western Blotting gezeigt werden konnte. Dies legt nahe, dass die AMPK-Aktivität die Membrantranslokation von SGLT1 erhöhen könnte. Diese Beobachtungen deuten auf eine Rolle der AMPK für die Regulation des Na+- gekoppelten Glukosetransports. Die Glutamattransporter EAAT3 und EAAT4 werden in Neuronen exprimiert. Durch die Aufnahme von Glutamat und Aspartat in die Neuronen dienen die beiden Transporter der Entfernung anregender Transmitter aus dem Extrazellulärraum. In einer Sauerstoffmangelsituation sammelt sich Glutamat im Extrazellulärraum an und kann damit exzitotoxisch wirken. Diese Studie erforschte die Möglichkeit der Regulation von EAAT3 und/oder EAAT4 durch die AMPK. Hierzu wurde die cRNA von EAAT3 oder EAAT4 sowohl mit als auch ohne cRNA der AMPK in Xenopus Oozyten exprimiert. Der elektrogene Na+/Glutamat Kotransport wurde elektrophysiologisch gemessen. In EAAT3- und in EAAT4- exprimierenden Oozyten generierte Glutamat einen Strom (Ig), welcher seine halbmaximale Stärke bei Glutamatkonzentrationen von (KM EAAT3) = 74µM (EAAT3) beziehungsweise (KM EAAT4) = 4µM (EAAT4) erreichte. Die Koexpression der konstitutiv aktiven gammaR70QAMPK oder der Wildtyp-AMPK hatte keinen Einfluss auf KM, aber reduzierte den maximalen Strom Ig: in Oozyten mit EAAT3 um 34% und Oozyten mit EAAT4 um 49%. Die Koexpression der inaktiven Mutante alphaK45RAMPK (alpha1 (K45R) beta1gamma1) hatte keinen erkennbaren Einfluss auf Ig. Unter dem Einfluss von gammaR70QAMPK oder des Wildtyps der AMPK wurden deutlich weniger EAAT3- und EAAT4-Transporter in die Membran eingebaut. Dies wurde mit Konfokalmikroskopie und Chemilumineszenz nachgewiesen. Die Untersuchungen zeigten, dass die AMPK den Na+-gekoppelten Glutamat Transport reduziert. AMP-activated protein kinase (AMPK), an energy-sensing enzyme, counteracts energy depletion by stimulation of energy production and limitation of energy utilization. On energy depletion, erythrocytes undergo suicidal death or eryptosis, triggered by an increase in cytosolic Ca2+ activity ([Ca2+]i) and characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. The present study explored whether AMPK participates in the regulation of eryptosis. Western blotting and confocal microscopy disclosed AMPK expression in erythrocytes. [Ca2+]i (Fluo3 fluorescence), cell volume (forward scatter), and PS exposure (annexin V binding) were determined by fluorescence-activated cell sorting (FACS) analysis. Glucose removal increased [Ca2+]i, decreased cell volume, and increased PS exposure. The AMPK-inhibitor compound C (20 myM) did not significantly modify eryptosis under glucose-replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca2+]i by Ca2+ ionophore ionomycin triggered eryptosis, an effect blunted by the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mM). As compared with erythrocytes from wild-type littermates (ampk+/+), erythrocytes from AMPKalpha1-deficient mice (ampk-/-) were significantly more susceptible to the eryptotic effect of energy depletion. The ampk-/- mice were anemic despite excessive reticulocytosis, and they suffered from severe splenomegaly, again pointing to enhanced erythrocyte turnover. The observations disclose a critical role of AMPK in the survival of circulating erythrocytes. AMP-activated protein kinase, a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilisation. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na+-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active gammaR70QAMPK (alpha1beta1gamma1 (R70Q)) alone, the addition of glucose to the extracellular bath generated a current (Ig), which was halfmaximal (KM) at = 650 µM glucose concentration. Coexpression of gammaR70QAMPK did not affect KM but significantly enhanced the maximal current (=1.7 fold). Coexpression of wild type AMPK or the kinase dead alphaK45RAMPK mutant (alpha1 (K45R) beta1gamma1) did not appreciably affect Ig. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 µM) enhanced the SGLT1 protein abundance in the cell membrane of Caco-2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na+-coupled glucose transport. The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase, a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4- expressing oocytes glutamate generated a current (Ig), which was half maximal (KM) at 74 µM (EAAT3) or at 4 µM (EAAT4) glutamate. Coexpression of constitutively active gammaR70QAMPK or of wild type AMPK did not affect KM but significantly decreased the maximal Ig in both EAAT3-(by 34%) and EAAT4-(by 49%) expressing oocytes. Coexpression of the inactive mutant alphaK45R AMPK (alpha1 (K45R) beta1gamma1) did not appreciably affect Ig. According to confocal microscopy and chemiluminescence coexpression of gammaR70QAMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK downregulates Na+-coupled glutamate transport.

Die Bewegungs-kontrollstrategien kontextabhängig und abhängig von unterschiedlichen Kriterien ausgewählt werden. Einerseits ist die Stabilität in den Bewegungszuständen wie der Fortbewegung ausschlaggebend für die ungestörte Ausführung bestimmter Handlungen und erfordert eine effektive Steuerung durch das zentrale Nervensystem. Andererseits wird die Bewegungsstrategieauswahl durch das zentrale Nervensystem dadurch bestimmt, dass die Energiekosten minimiert werden soll. Beide Konzepte (d.h. die Aufrechterhaltung der Stabilität und die Energiekostenminimierung) spielen eine fundamentale Rolle bei der Frage, warum sich Menschen so bewegen, wie sie es tun. Unklar ist dabei allerdings, auf welche Weise das zentrale Nervensystem beide Prinzipien gegeneinander gewichtet. In den letzten 20 Jahren haben uns wissenschaftliche Konzepte wie die Chaostheorie oder die Theorie komplexer Systeme eine neue Herangehensweise an diese Fragen ermöglicht. Diese Arbeit untersucht die dynamische Stabilität menschlicher Fortbewegung mit Hilfe des Konzepts der Ljapunowanalyse. Als erstes wird eine methodologische Untersuchung der Verlässlichkeit des maximalen Ljapunowexponenten beim Gehen und Laufen durchgeführt (Kapitel 2). Danach wird verglichen zwischen dem Laufen unter normalen Umständen und dem darauffolgenden Laufen ohne Schuhe, wobei letzteres eine Abnahme der Stabilität nach dem Übergang zu den neuen Umständen zur Folge hat (Kapitel 3). In der letzten Untersuchung wurde ein unterschiedlich langes Training zur Verbesserung der Laufenergetik durchgeführt, in einer Gruppe nur über einen kurzen und in einer anderen Gruppe über einen etwas längeren Zeitraum (Kapitel 4). Die Ergebnisse zeigen, dass Bewegungskontrollfehler für die Energiekosten beim Laufen eine Rolle spielen können, und legen somit eine flexible Priorisierung der Bewegungskontrolle nahe. Motor control strategies are chosen in a context dependent manner, based on different criteria. On the one hand stability in dynamic conditions such as locomotion, is crucial to uninterrupted task execution and requires effective regulation by the central nervous system. On the other, minimization of the energetic cost of transport is instrumental in choosing the locomotion strategy by the central nervous system. Both these concepts, (i.e. maintaining stability and optimization of energetic cost of locomotion) have a fundamental role on how and why humans move in the way they do. However, how the human central nervous system prioritizes between the different goals is unknown. In the last 20 years, ideas from scientific paradigms such as chaos theory and complex systems have given us novel tools to approach these questions. The current thesis examines the dynamic stability during human locomotion under such an approach using the concept of Lyapunov analysis. At first a methodological examination of the reliability of the maximum Lyapunov exponent in walking and running has been conducted (chapter 2). Afterwards, an examination between the habitual running condition and after removal of footwear was conducted, exhibiting a decrease in stability following the acute transition to the new condition (chapter 3). In the last study, a training intervention aiming at improvements in running energetics was performed using a short-term and a long-term intervention group (chapter 4). The results evidence that motor control errors can have a role in the energy cost of running and thus, a flexible prioritization of the motor control output.

CYP2D6 metabolizes a range of centrally acting drugs, neurotoxins, and endogenous neurochemicals. Higher levels of brain, but not liver, CYP2D6 have been identified in alcoholics and smokers, suggesting exposure to ethanol and/or nicotine may induce brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self-administration and nicotine treatment on CYP2D expression. METHODS: Monkeys were randomized into 4 groups of 10/group consisting of a control group, ethanol-only group, nicotine-only group, and a combined ethanol and nicotine group; treatments occurred for 64 days. RESULTS: Exposure to chronic ethanol and nicotine induced CYP2D across various brain regions and cell types, particularly when both drugs were given in combination. No changes in protein levels were observed in liver or in CYP2D mRNA levels in liver and brain. CONCLUSIONS: Ethanol and nicotine increase brain CYP2D levels, which may affect CNS drug response, neurodegeneration and personality among those exposed to alcohol and/or nicotine. ; MAST

High performance gradient and shim coils are highly interested for high-field magnetic resonance imaging and spectroscopy to correct for large B0 inhomogeneities created by the magnetic susceptibility differences between tissues, bone, and air. In chapter two, complete sets of high-performance gradient and shim coils are designed using two different methods: the minimum inductance and the minimum power target field methods. A quantitative comparison of shim performance in terms of merit of inductance, ML, and merit of resistance, MR, is made for shim coils designed using the minimum inductance and the minimum power design algorithms. The coils designed using the target field method are not controlled over the length of the coil. In order to produce realistic coils for use in human or small-animal studies, direct control over the length of the coils is necessary. Therefore in chapter three, an extended Fourier series method for the design of shim coils with predetermined length is presented. This simple method is based on a truncated Fourier series expansion of the current density to allow for explicit control over the coil length. This method is mathematically simple, easy to implement and computationally fast. Also a quantitative comparison of figures of merit for inductance and resistance is made as a function of shim coil length. Coils of 40 cm diameter are designed with lengths of 50 cm, 60 cm, 80 cm, and 100 cm. Pushing the boundaries of shim design in MRI, we designed a region specific, custom shim coil to correct for large field inhomogeneities that are consistent among subjects. In chapter four, we have designed a custom shim coil for the medial temporal lobe of the human head to correct for the significant field inhomogeneities caused by magnetic susceptibility differences at air/tissue interfaces. The custom coil was designed using the boundary element method. This method is capable of designing coils wound on arbitrarily shaped surfaces so as to produce specific field shapes. We propose that, the ...

Background: Obesity and metabolic syndrome are highly complex disease states and still needs effective treatment and prevention strategies. Growing evidence suggests that dietary milk proteins and prebiotics plays a role in preventing metabolic disorders; however, the underlying mechanisms are unknown. Objective: This dissertation examines how dietary milk proteins and prebiotics (inulin fiber) affect energy balance, host physiology, and gut microbiota to affect metabolic health. The overall objectives of this thesis include: 1) assess the effects of milk protein components on energy balance and stroke onset in stroke-prone rats; 2) determine the role of prebiotics and gut microbiota in regulation of energy balance in obesity-prone and obesity-resistant rats; 3) assess the combined effects of milk protein components and prebiotic on energy balance in obese rats. Methods: Animal studies were conducted using male wistar-kyoto, spontaneously hypertensive stroke-prone, sprague-dawley (SD), obesity-prone (OP) and obesity-resistant(OR) rats. Energy intake, meal patterns, respiratory quotient, and energy expenditure were measured using CLAMS metabolic chambers. Body composition was measured with magnetic resonance imaging. Intraperitoneal glucose and meal tolerance tests were conducted to measure glucose and plasma hormone concentrations. Gut microbiota was assessed using qPCR and 16S rRNA gene sequencing. Gene mRNA abundance was measured using real-time RT-PCR. Results: The primary findings from our study objectives were: 1) supplementation of dietary casein, whey, or its components lactalbumin and lactoferrin, improved energy balance, prevented neurological deficits, morbidity and renal damage and delayed the onset of stroke in stroke-prone rats; 2) gut microbiota play an indispensable role in mediating prebiotic fiber-induced satiety via its effects on cholecystokinin-A and peptide YY Y-2 receptor signaling in high-fat-fed SD, OP, and OR rats; 3) combination of lactoferrin and inulin additively improved energy balance and decreased body weight and adiposity in diet-induced obese rats. Conclusion: Our results provide evidence for the role of milk protein components and prebiotics in improving metabolic dysfunctions in obesity and metabolic syndrome. The findings from our preclinical studies provide a rationale for clinical trials assessing the effects of milk protein components and prebiotics in the prevention and treatment of obesity and its related metabolic abnormalities.

Arthroscopic surgery is a type of Minimally Invasive Surgery (MIS) performed in human joints, which can be used for diagnostic or treatment purposes. The nature of this type of surgery makes it such that surgeons require extensive training to become experts at performing surgical tasks in tight environments and with reduced force feedback. MIS increases the possibility of erroneous actions, which could result in injury to the patient. Many of these injuries can be prevented by implementing appropriate training and skills assessment methods. Various performance methods, including Global Rating Scales and technical measures, have been proposed in the literature. However, there is still a need to further improve the accuracy of surgical skills assessment and improve its ability to distinguish fine variations in surgical proficiency. The main goal of this thesis is to enhance surgical, and specifically, arthroscopic skills assessment. The optimal assessment method should be objective, distinguish between subjects with different levels of expertise, and be computationally efficient. This thesis proposes a new method of investigating surgical skills by introducing energy expenditure metrics. To this end, two main approaches are pursued: 1) evaluating the kinematics of instrument motion, and 2) exploring the muscle activity of trainees. Mechanical energy expenditure and work are investigated for a variety of laparoscopic and arthroscopic tasks. The results obtained in this thesis demonstrate that expert surgeons expend less energy than novice trainees. The different forms of mechanical energy expenditure were combined through optimization methods and machine learning algorithms. An optimum two-step optimization method for classifying trainees into detailed levels of expertise is proposed that demonstrates an enhanced ability to determine the level of expertise of trainees compared to other published methods. Furthermore, performance metrics are proposed based on electromyography signals of the forearm muscles, which ...

AMP-activated protein kinase (AMPK), an energy-sensing enzyme, counteracts energy depletion by stimulation of energy production and limitation of energy utilization. On energy depletion, erythrocytes undergo suicidal death or eryptosis, triggered by an increase in cytosolic Ca2+ activity ([Ca2+]i) and characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. The present study explored whether AMPK participates in the regulation of eryptosis. Western blotting and confocal microscopy disclosed AMPK expression in erythrocytes. [Ca2+]i (Fluo3 fluorescence), cell volume (forward scatter), and PS exposure (annexin V binding) were determined by fluorescence-activated cell sorting (FACS) analysis. Glucose removal increased [Ca2+]i, decreased cell volume, and increased PS exposure. The AMPK-inhibitor compound C (20 myM) did not significantly modify eryptosis under glucose-replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca2+]i by Ca2+ ionophore ionomycin triggered eryptosis, an effect blunted by the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mM). As compared with erythrocytes from wild-type littermates (ampk+/+), erythrocytes from AMPKalpha1-deficient mice (ampk-/-) were significantly more susceptible to the eryptotic effect of energy depletion. The ampk-/- mice were anemic despite excessive reticulocytosis, and they suffered from severe splenomegaly, again pointing to enhanced erythrocyte turnover. The observations disclose a critical role of AMPK in the survival of circulating erythrocytes. AMP-activated protein kinase, a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilisation. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na+-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active gammaR70QAMPK (alpha1beta1gamma1 (R70Q)) alone, the addition of glucose to the extracellular bath generated a current (Ig), which was halfmaximal (KM) at = 650 µM glucose concentration. Coexpression of gammaR70QAMPK did not affect KM but significantly enhanced the maximal current (=1.7 fold). Coexpression of wild type AMPK or the kinase dead alphaK45RAMPK mutant (alpha1 (K45R) beta1gamma1) did not appreciably affect Ig. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 µM) enhanced the SGLT1 protein abundance in the cell membrane of Caco-2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na+-coupled glucose transport. The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase, a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4- expressing oocytes glutamate generated a current (Ig), which was half maximal (KM) at 74 µM (EAAT3) or at 4 µM (EAAT4) glutamate. Coexpression of constitutively active gammaR70QAMPK or of wild type AMPK did not affect KM but significantly decreased the maximal Ig in both EAAT3-(by 34%) and EAAT4-(by 49%) expressing oocytes. Coexpression of the inactive mutant alphaK45R AMPK (alpha1 (K45R) beta1gamma1) did not appreciably affect Ig. According to confocal microscopy and chemiluminescence coexpression of gammaR70QAMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK downregulates Na+-coupled glutamate transport. ; Die AMP-aktivierte Proteinkinase (AMPK) ist ein Enzym, das den Energiehaushalt der Zelle reguliert: AMPK erhöht die intrazelluläre ATP Konzentration, indem es energieverbrauchende Prozesse bremst und energieproduzierende fördert. Im Falle erschöpfter Energiereserven starten Erythrozyten ihr Apoptose-Programm, auch Eryptose genannt. Initiiert durch eine Zunahme der zytosolischen Ca2+-Konzentration ([Ca2+]i) kommt es zu Zellschrumpfung und der Exposition von Phosphatidylserin (PS) auf der Außenseite der Erythrozytenmembran. Die vorliegende Studie untersuchte, welche Rolle die AMPK in der Regulation der Eryptosis spielt. Die Expression von AMPK in den Erythrocyten wurde mittels der Western Blot Methode und Konfokalmikroskopie nachgewiesen. Die intrazelluläre Ca2+-Konzentration ([Ca2+]i), das Zellvolumen der Erythrozyten sowie Phosphatidylserin auf der Außenseite der Membran wurden mittels Durchflusszytometrie (FACS) ermittelt. Glukosemangel im Extrazellulärmedium führte zu einer Erhöhung von [Ca2+]i, verkleinerte das Zellvolumen und erhöhte den Anteil von PS in der äußeren Membran. Versuchsreihen mit dem AMPK-Inhibitor Compound C (20 µM) ergaben, dass bei Anwesenheit von Glukose keine signifikante Veränderung der Eryptoserate eintritt. Hingegen verstärkte Compound C bei Glucoseentzug die Eryptose deutlich. Das Ca2+-Ionophor Ionomycin führt zu einer Erhöhung von [Ca2+]i, welche wiederum die Eryptose auslöst. Dieser Effekt wurde durch den AMPK-Aktivator 5-Aminoimidazol-4-carboxamid-1-beta-D-ribofuranosid (AICAR; 1 mM) abgeschwächt. Verglichen mit Erythrozyten des Wild-Typs (ampk+/+), waren Erythrozyten von AMPK&alpha1- defizienten Mäusen (ampk-/-) signifikant anfälliger für Energiemangel bedingte Eryptose. Die ampk-/--Mäuse hatten trotz exzessiver Retikulozytose eine Anämie und litten unter schwerer Splenomegalie, was auf einen erhöhten Erythrozytenumsatz hinweist. Diese Beobachtungen zeigen, dass AMPK die Lebensdauer von zirkulierenden Erythrozyten beeinflusst. AMP-aktivierte Protein Kinase, eine Serin/Threonin-Kinase, wird durch Energiemangel aktiviert. Sie stimuliert die Energieprodukion und hemmt den Energieverbrauch. Dieser Effekt der AMPK wurde insofern für die GLUT-Familie der erleichterten Glucosetransporter nachgewiesen, als die Aktivierung der AMPK zu einem erhöhten Glukoseeinstrom druch GLUT-Transporter führt. Die vorliegende Studie untersuchte die Möglichkeit, ob die AMPK eventuell den Na+ gekoppelten Glukosetransport durch SGLT1 (SLC5A1) reguliert. Dazu wurde die cRNA von SGLT1 in Xenopus laevis-Oozyten mit und ohne AMPK exprimiert. Der elektrogene Symport von Na+/Glucose wurde mithilfe der Zwei-Elektroden-Spannungsklemme (DEVC) gemessen. In SGLT1- exprimierenden Oozyten konnte durch die Zugabe von Glukose zum extrazellulären Bad ein Strom (Ig) generiert werden. Dieser erreichte seine halbmaximale Stärke bei einer Konzentration von (KM) = 650 µM Glucose. Bei Oozyten, in die lediglich Wasser oder die konstitutiv aktive gammaR70QAMPK (alpha1beta1gamma1(R70Q)) injiziert wurde, ließ sich dieser Strom durch Glukosezugabe nicht erzeugen. Die Koexpression von gammaR70QAMPK hatte keinen Einfluss auf KM, erhöhte aber signifikant den maximalen Strom Ig max (= 1.7 fach). Die Koexpression des AMPK-Wildtyps oder der inaktivierten alphaK45RAMPK Mutante (alpha1(K45R)beta1gamma1) wirkte sich auf Ig nicht messbar aus. AICAR (1 mM), Phenformin (1 mM) und A-769662 (10 µM) erhöhten die SGLT1-Expression in der Zellmembran von Caco-2 Zellen, wie durch Konfokalmikroskopie und Western Blotting gezeigt werden konnte. Dies legt nahe, dass die AMPK-Aktivität die Membrantranslokation von SGLT1 erhöhen könnte. Diese Beobachtungen deuten auf eine Rolle der AMPK für die Regulation des Na+- gekoppelten Glukosetransports. Die Glutamattransporter EAAT3 und EAAT4 werden in Neuronen exprimiert. Durch die Aufnahme von Glutamat und Aspartat in die Neuronen dienen die beiden Transporter der Entfernung anregender Transmitter aus dem Extrazellulärraum. In einer Sauerstoffmangelsituation sammelt sich Glutamat im Extrazellulärraum an und kann damit exzitotoxisch wirken. Diese Studie erforschte die Möglichkeit der Regulation von EAAT3 und/oder EAAT4 durch die AMPK. Hierzu wurde die cRNA von EAAT3 oder EAAT4 sowohl mit als auch ohne cRNA der AMPK in Xenopus Oozyten exprimiert. Der elektrogene Na+/Glutamat Kotransport wurde elektrophysiologisch gemessen. In EAAT3- und in EAAT4- exprimierenden Oozyten generierte Glutamat einen Strom (Ig), welcher seine halbmaximale Stärke bei Glutamatkonzentrationen von (KM EAAT3) = 74µM (EAAT3) beziehungsweise (KM EAAT4) = 4µM (EAAT4) erreichte. Die Koexpression der konstitutiv aktiven gammaR70QAMPK oder der Wildtyp-AMPK hatte keinen Einfluss auf KM, aber reduzierte den maximalen Strom Ig: in Oozyten mit EAAT3 um 34% und Oozyten mit EAAT4 um 49%. Die Koexpression der inaktiven Mutante alphaK45RAMPK (alpha1 (K45R) beta1gamma1) hatte keinen erkennbaren Einfluss auf Ig. Unter dem Einfluss von gammaR70QAMPK oder des Wildtyps der AMPK wurden deutlich weniger EAAT3- und EAAT4-Transporter in die Membran eingebaut. Dies wurde mit Konfokalmikroskopie und Chemilumineszenz nachgewiesen. Die Untersuchungen zeigten, dass die AMPK den Na+-gekoppelten Glutamat Transport reduziert.

Objectives: Most weight loss medications target reductions in energy intake while neglecting energy expenditure, a critical predictor of weight loss/regain. This pilot study examined the effect of short-acting methylphenidate (MPH) on resting energy expenditure (REE), thermic effect of food (TEF), physical activity energy expenditure (PAEE), and how changes in energy expenditure relate to changes in body composition in youth and adults living with obesity. Methods: This study was a randomized, double-blind, placebo-controlled two-parallel arm study. In total, 19 participants were screened, of which 14 participants were randomized into the study, but complete data was only collected for 12, and only analyzed for 10 participants. Those 10 participants aged 28.8 ± 6.9 yrs. (5 Male, 5 Female) were randomized to receive either MPH (0.5 mg/kg) (n = 5) or placebo (n =5) twice daily for 60 days. Participants’ REE and TEF (indirect calorimetry), were measured at baseline (no drug/placebo), and day 60 post-treatment (drug/placebo). Participants’ PAEE (Actical) was measured between screening and baseline for a 1-week period (no drug/placebo), and on day 53 for a 1-week period (drug/placebo). Participants’ anthropometrics were measured using DEXA at baseline, and day 60 post-treatment. Results: From baseline to day 60, MPH showed a relative difference to placebo in relative REE (Relative REE: F(1, 8) = 4.235, p = 0.074, d = 0.83, 2 = 0.346) of 10%, evidenced by a 6% increase in relative REE kcal/kg (18.53 ± 1.97 Kcal/day/kg at baseline, 19.71 ± 2.52 Kcal/day/kg at final) for the MPH group, and a 4% decrease (19.08 2.36 Kcal/day/kg at baseline, 18.26 ± 2.04 Kcal/day/kg at final) in placebo, translating to moderate-effect size (Cohen’s d=0.63) favouring MPH. From baseline to day 60, there were no significant differences between groups on changes in TEF (TEF AUC: F(1, 8) = 0.079, p = 0.785, d = 0.15, 2 = 0.010) or any PAEE variables such as sedentary behavior (SB: F (1, 8) = 0.455, p = 0.52, d = 0.02, 2 = 0.054), light physical activity (LPA: F (1, 8) = 0.504, p = 0.50, d = 0.16, 2 = 0.059), moderate physical activity (MPA: F (1, 8) = 0.281, p = 0.61, d = 0.19, 2 = 0.034), moderate-to-vigorous physical activity (MVPA: F (1, 8) = 0.120, p = 0.74, d = 0.15, 2 = 0.015), or vigorous physical activity (VPA: F (1, 8) = 3.495, p = 0.098, d = 0.91, 2 = 0.304) . Mean change in body weight (kg) resulted in a weight loss of roughly -2.66 ± 2.00 kg in the MPH group and -1.64 ± 1.41 kg in the placebo group, differences that were not statistically significant. Mean change in both groups for body fat% of -0.33 ± 2.08 %, mean change in fat mass of -1.05 ± 2.59 kg, and finally a mean change in fat-free mass of -0.06 ± 1.19 kg was reported. Changes in relative REE were inversely correlated with changes in body weight (r = -0.599, p = 0.067), body fat (r = -0.524, p = 0.12) and fat mass (r = -0.599, p = 0.096). These associations were stronger in the MPH group. Conclusions: Our data suggests that MPH administration may lead to a meaningful increase in relative REE, and these suggested changes were associated with reductions in adiposity among individuals with obesity. These preliminary findings suggest that MPH should be further examined using a larger sample size and study duration to determine its effectiveness in promoting weight loss and maintenance of weight loss in individuals with obesity, a population at high risk of morbidity and premature mortality.