Stress is defined as a challenge to homeostatic equilibrium by physical or psychological events, generating a coping response consisting of central and peripheral changes, with the aim of exerting control over the threatening events. Corticotropin-releasing factor (CRF) is well known as a hypothalamic factor which controls the hypothalamic-pituitary-adrenocortical (HPA) axis during basal activity and stress. CRF also serves a neurotransmitter function in the brain, where it is implicated in a range of stress-related behaviours. The measurement of local cerebral glucose utilisation (LCGU) using radiolabelled 2-deoxy-D-glucose (2DG) provides an estimate of cellular activity in the brain. 2DG competes with glucose in its metabolic pathway, but is not fully metabolised, instead accumulating within astrocytes where it can be quantified. After consideration of available modifications to the LCGU technique, the effect of manipulating the CRF system on LCGU was studied, in order to test the hypothesis that CRF and other endogenously expressed CRF-related peptides would induce different patterns of LCGU, and to examine the involvement of CRF receptors in any response. The CRF1 receptor has been implicated in the mediation of stress- and anxiety-related behaviour, while recent evidence has suggested a role for CRF2 in mediating the delayed effects of stress, although it has previously been postulated that CRF2 may be involved in the attenuation of stress-related behaviour. CRF and the endogenous CRF-related peptide Urocortin 1 both induced increases in LCGU in a number of brain regions associated with the CRF system, with concomitant activation of the HPA axis. CRF induced increases in LCGU in the dissected hypothalamus, thalamus, cerebellum and hippocampus, while Urocortin 1 induced a significant increase in LCGU in a dissected hindbrain region, with trend-like effects in frontal cortex and hippocampus. These regions contain components of the CRF system, or receive projections from regions involved in the CRF system, and have been implicated in stress-related function. The effects of CRF on LCGU appear to be mediated by the CRF2 receptor, as they were abolished by the selective CRF2 antagonist antisauvagine-30, but persisted in mice lacking CRF1 and were unaffected by a selective CRF1 antagonist. However, neither of the endogenous CRF-related peptides selective for CRF2, Urocortin 2 and Stresscopin, affected LCGU, which may indicate ligand-specific effects within the CRF system. In contrast to the effects of CRF, restraint stress reduced LCGU, while activating the HPA axis, and this response was unaffected by a selective CRF1 antagonist. This data suggests that the role of CRF receptors in restraint-induced LCGU changes may be overshadowed by effects on other neurotransmitter systems. These studies support the hypothesis that CRF and other endogenously expressed CRF-related peptides would induce different patterns of LCGU, and highlight the involvement of particular brain regions in the response to CRF receptor stimulation. Furthermore, these studies provide evidence suggesting ligand-specific effects within the CRF system.

Osteoblasts are bone-forming cells and therefore they are responsible of the synthesis of type I collagen, the main component of bone matrix. However, there is an apparent disconnect between the regulation of osteoblast differentiation and bone formation since the synthesis of Type I collagen precedes the expression of Runx2, the earliest determinant of osteoblast differentiation. Recently, genetic experiments in the mouse have revealed the existence of an unexpected cross-regulation between bone and other organs. In particular this body of work has highlighted the importance of osteoblasts as endocrine cells to regulate whole-body glucose homeostasis by secretion of a hormone, osteocalcin. However, the fundamental question of why bone regulates glucose homeostasis remained to be answered. Therefore, in my thesis, considering that bone is a metabolically demanding organ that constantly renews itself, I hypothesized that characterizing the connection between the need of glucose as a main nutrient in osteoblasts and bone development will provide a key to deeper understanding of why bone regulates glucose homeostasis. My work shows here that glucose uptake through GLUT1 in osteoblasts is needed for osteoblast differentiation by suppressing the AMPK-dependent activation by phosphorylation at S148 of Smurf1 that targets Runx2 for degradation. I also uncovered the mechanism of action of Smurf1 in this setting. In a distinct but synergetic way, glucose uptake promotes bone formation by inhibiting a distinct function of AMPK. In turn, Runx2 favors Glut1 expression, and this feedforward regulation between Runx2 and Glut1 determines the onset of osteoblast differentiation during development and the extent of bone formation throughout life. Furthermore, I also identified that Smurf1 not only regulates osteoblast differentiation by targeting Runx2 for degradation but also contributes to whole-body glucose homeostasis by regulating the activation of osteocalcin by targeting the insulin receptor for degradation in vivo. These results identify Smurf1 as a determinant of osteoblast differentiation during development, of bone formation and glucose homeostasis post-natally. Most importantly, we show that these Smurf1 functions required AMPK-phosphorylation site S148 in vivo. Altogether, these results revealed the absolute necessity of glucose as a regulator of Runx2 accumulation during osteoblast differentiation and bone formation in vivo and highlight the fundamental importance of the intricate cross-talk between bone and whole-body glucose metabolism.

Thesis. 1978. M.S.--Massachusetts Institute of Technology. Dept. of Chemical Engineering. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 253-265. / M.S.

Alzheimer’s disease (AD) is the most common cause of dementia, accounting for around 60-70% of cases. AD encompasses large-scale neuronal loss, resulting in progressive memory and other cognitive decline. Presently, there is no cure for dementia and in light of the ageing population demographic, this represents a clear unmet medical and socioeconomic challenge Worldwide. Much of the current AD research focuses on studying the brain once hallmark amyloid plaque and neurofibrillary tangle pathologies have presented. However their appearance is extremely end stage and to date, any therapeutic interventions aimed at alleviating them having failed to halt symptoms progression. It may therefore be beneficial to look for earlier changes, with metabolic and oxidative stress events as well as reduced cerebral metabolism thought to occur early on in disease progression. Evidence from rare, familial AD cases suggests a causative role for A in AD pathogenesis. For this reason, the enzyme beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), the rate-limiting step in A production is currently of great therapeutic interest. With the prevailing view being that reducing BACE1 levels will be beneficial in AD, there remains a need to better understand the physiological roles of BACE1 to avoid potential side effects of BACE1 inhibition. Herein is presented data showing that, in agreement with the previous literature, BACE1 is fundamentally regulated by cell stress. Notably, both acute and prolonged bouts of oxidative and metabolic stress result in significant increases in BACE1 and APP protein expression. These changes also result in a shift in APP metabolism, with amyloidogenic processing of APP predominating during times of stress. It has also been shown that chronic elevation of BACE1 and/or manipulation of APP processing can alter cellular glucose uptake and use. These changes were determined through the use of radiolabelled substrate uptake and oxidation as well as extracellular flux assays. These data highlighted a fundamental shift in cellular metabolism, with aerobic glycolysis being utilised over oxidative metabolism of glucose. These changes were later shown to come as a result of metabolic lesions, which acted to impair substrate delivery to the electron transport chain of the mitochondria. Taken together, these data show that overexpression of the AD-associated protein BACE1 phenocopies a number of the earliest detectable changes observed in the brains of people who later develop AD. Finally, these data highlighted the potential importance of a number of novel pathways (Sirtuin, AMP-activated protein kinase, and peroxisome proliferator-activated receptor- coactivator signalling) that may underlie these changes and offer therapeutic avenues for earlier and more targeted treatment to halt AD progression.

The insulin-dependent uptake of glucose by adipose and muscle tissues is accomplished through the regulated vesicle trafficking of the GLUT4 glucose transporter to the plasma membrane. The distal trafficking events comprising the tethering, docking and fusion of GLUT4 vesicles with the plasma membrane are poorly defined, but represent vital steps in this pathway. This dissertation encompasses a series of complementary studies that have provided new insights into how these events are regulated in the adipocyte. The Sec1p homologue Munc18c, is believed to play a central role in the docking of GLUT4 vesicles by controlling SNARE complex assembly. Munc18c was shown to bind the t-SNARE Syntaxin4 and form a stable complex in vivo. Protein binding studies demonstrated that Munc18c interacts with Syntaxin4 via an evolutionarily conserved N-terminal binding mode and the formation of the Munc18c/Syntaxin4 hetero-dimer was shown to promote SNARE complex assembly. In contrast to previous reports, I propose that Munc18c is positive regulator of SNARE assembly and vesicle docking. The exocyst complex is thought to promote the tethering of exocytic GLUT4 vesicles with the plasma membrane. Yeast two-hybrid screens revealed interactions between the exocyst subunits Sec6 and Exo70 and the SNARE-associated proteins Munc18c and Snapin, respectively. Snapin was subsequently shown to have a novel role in GLUT4 trafficking. These interactions suggest Munc18c and Snapin provide a course for cross-talk between the exocyst complex and the SNAREs to stimulate GLUT4 vesicle tethering and docking. In addition to its interactions with Munc18c and Snapin, the exocyst was also found to interact with the GTP-bound form of RalA, a small GTPase regulated by insulin. RalA was almost exclusively localised to the plasma membrane of the adipocyte and a novel role for the RalA/exocyst interaction in GLUT4 trafficking was demonstrated. Specifically, overexpression of a GTP-deficient RalA mutant significantly inhibited insulin-stimulated GLUT4 appearance on the plasma membrane. In addition to its role in GLUT4 trafficking, a novel role for RalA was demonstrated in insulin release from pancreatic -cells, indicating that RalA may represent a universal component of regulated exocytosis. It is becoming increasingly apparent that vesicle trafficking events from yeast to mammals rely on similar protein complexes which communicate through multiple protein interactions, ensuring vesicle transport is highly coupled. Similarly, the Munc18c studies demonstrate that while mammalian cells have evolved to fulfil specialised functions throughout the body, some proteins appear to have retained the biochemical properties of their ancestors, emphasing the importance of this family of proteins throughout eukaryotic vesicle transport. In contrast, proteins such as RalA have evolved only in higher eukaryotes and appear to play a universal role in vesicle transport despite vast differences in the specialised functioning of mammalian cells.

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This study examined the effects of exercise on glucose metabolism in adipocytes from female rats. Female rats were exercised by swimming six hours per day, five days per week for eight weeks. There was no variation in body weight gain (P > 0.05) between the exercise and control animals through the experimental period. The swimmers' fat cells were smaller (P < 0.05) than those of the sedentary controls of the same age. The rates of glucose oxidation of both C-1 and C-6 glucose were significantly higher (P < 0.05) in the exercise rats' adipocytes at all insulin concentrations. The sedentary control rats' adipocytes showed no significant response at any insulin concentration. Thus, exercise is a significant stimulus to cause increased oxidation rates in the adipocytes from exercising rats. These data also indicate that glucose transport, not defective glucose oxidation, is the limiting mechanism that accounts for the decreased responsiveness of adipocytes from sedentary control animals.

The purpose of this investigation was to determine the relationship between calcium and glucose uptake following muscle contraction with the use of the calcium channel blocker dantrolene. In previous studies an exercise model has been used to investigate the role of calcium during post-exercise glucose uptake. This study utilized electrical stimulation. It has been shown that exercise-induced glucose uptake is calciummediated, but to date no one has shown that glucose transport induced by electrical stimulation is calcium-mediated. Twenty four male Sprague Dawley rats weighing 140 g were sacrificed and their epitrochlearis muscles were removed. Four treatment groups were established: control, muscle incubated in glucose (4mM); insulin, muscles incubated in glucose (4mM) and insulin (1000uU/ml); electrical stimulation, at 50 Hz for two five minute intervals separated by one minute rest periods; insulin (1000uU/ml) and electrical stimulation at 50 Hz for two five minute intervals separated by one minute intervals. Each group consisted of contain 8-10 muscle preparations. Glucose uptake was measured through the use of a double label of radioactive mannitol and 3-O-methylglucose and analyzed using liquid scintillation. This project followed a randomized group design. Treatments were measured with a one way ANOVA. / School of Physical Education

No description available.