The integrated physiology of glucose homeostasis: regulation by extracellular and intracellular nucleotide sensors

Physiological glucose levels are maintained by the complex integration of neuroendocrine, hormonal and nutritional signals controlled by multiple tissues in the body. A dysregulation in these mechanisms leads to increasingly prevalent conditions characterised by an inability to regulate blood glucose levels, such as diabetes. Maintaining glycaemia within a target range remains a daily challenge for individuals with both Type 1 and Type 2 diabetes and a better understanding of the pathophysiology of impaired glucose homeostasis in these conditions is still required to identify more effective and targeted therapeutic approaches. Work in this thesis focused on elucidating the mechanisms by which lipid overflow, be it from increasingly sedentary behaviour or overfeeding, leads to the development of insulin and anabolic resistance in skeletal muscle. Loss of insulin-stimulated glucose clearance by skeletal muscle is a main driver for impaired glucose disposal in Type 2 diabetes and a role for excessive lipid availability in this pathology is well established. Here, muscle cells were treated with high concentrations of a saturated fatty acid and data demonstrated that lipid overflow led to impaired anabolic sensitivity, inflammatory cytokine release and mitochondrial dysfunction. Furthermore, these experiments elucidated a novel role for adenosine tri-phosphate, acting as a signalling molecule, in the regulation of muscle glucose metabolism, identifying insulin and exercise mimetic roles of the nucleotide that could be therapeutically targetable. This work was translated into humans, where the effect of lipid overflow by high-fat overfeeding was assessed in an experimental model of inactivity-induced insulin and anabolic resistance. Data suggested that two days of disuse (by forearm immobilisation) were sufficient to cause substantial muscle insulin resistance. After 7 days, muscle strength was significantly reduced and anabolic resistance was evident due to decreased forearm balance of potent anabolic amino acids such as leucine. Contrary to the hypothesis, high-fat overfeeding did not accelerate or exacerbate these impairments, suggesting that removal of contraction represents a potent stimulus for loss of substrate demand by muscle, irrespective of energy balance. Insulin replacement therapy has been the cornerstone of treatment for Type 1 and advanced Type 2 diabetes for over 8 decades. A serious and inadvertent consequence of prolonged insulin therapy is the increased risk of hypoglycaemia. Hypoglycaemia can lead to impaired physiological defences against a decrease in blood glucose and loss of awareness of these changes. AMP-activated protein kinase activators, which are widely used (to target peripheral tissues) as anti-hyperglycaemic agents in Type 2 diabetes have demonstrated central effects that amplify the first defence against hypoglycaemia, or counterregulatory response. Data presented here demonstrated that peripheral administration of a brain permeable AMP-activated protein kinase activator amplified the counterregulatory response to hypoglycaemia by enhancing glucagon levels in healthy rats, without altering fasting blood glucose. This demonstrates important clinical implications for the pharmaceutical use of AMP-activated protein kinase activators as the central roles that regulate blood glucose may supersede the peripheral effects of these compounds, during hypoglycaemia. Work presented here highlights the complexity of the regulation of glycaemia and discusses the contribution of extracellular and intracellular nucleotides/nucleotide sensors to glucose homeostasis. It can be concluded from this work that strategies to manage or treat diabetes in future should consider the importance of tissue-specific or metabolic status specific actions of the targets of interest.Diabetes UKJuvenile Diabetes Research Foundation Internationa

Solvation and surface effects on polymorph stabilities at the nanoscale

We explore the effects of particle size and solvent environment on the thermodynamic stability of two pairs of polymorphs subjected to ball-mill neat grinding (NG) and liquid assisted grinding (LAG). Two systems were studied: (i) forms I and II of a 1 : 1 theophylline : benzamide cocrystal and (ii) forms A and B of an aromatic disulfide compound. For both systems, the most stable-bulk polymorph converted to the metastable-bulk polymorph upon NG. LAG experiments yielded different outcomes depending on the amount of solvent used. This was further investigated by performing carefully controlled LAG experiments with increasing $\mu$L amounts of solvents of different nature. With these experiments, we were able to monitor form A to B and form I to II conversions as a function of solvent concentration and derive polymorph equilibrium curves. The concentration required for a switch in polymorphic outcome was found to be dependent on solvent nature. We propose that these experiments demonstrate a switch in thermodynamic stability of the polymorphs in the milling jar. Form B, the stable-bulk polymorph, has less stable surfaces than form A, thus becoming metastable at the nanoscale when surface effects become important. $\textit{Ex situ}$ diffraction and electron microscopy data confirm crystal sizes in the order of tens of nanometers after the ball mill grinding experiments reach equilibrium. DFT-d computations of the polymorph particles stabilities support these findings and were used to calculate cross-over sizes of forms A and B as a function of solvent. Attachment energies and surface stabilities of the various crystalline faces exposed were found to be very sensitive to the solvent environment. Our findings suggest that surface effects are significant in polymorphism at the nanoscale and that the outcomes of equilibrium ball-mill NG and LAG experiments are in general controlled by thermodynamics.Engineering and Physical Sciences Research Counci

Influence of channel aspect ratio on the onset of purely-elastic flow instabilities in three-dimensional planar cross-slots

In this work, we perform creeping-flow simulations of upper-convected Maxwell and simplified Phan-Thien-Tanner fluids to study the purely-elastic steady bifurcation and transition to time-dependent flow in three-dimensional planar cross-slots. By analysing the flow in geometries with aspect ratios ranging from the near Hele-Shaw flow like limit, up to the very deep, two-dimensional limit, we are able to characterize the mechanism of the cross-slot bifurcation with significant detail. We conclude that the bifurcation mechanism is similar to a buckling instability, by which fluid is redirected via paths of least resistance, resulting in the emergence of peripheral stagnation points, above and below the central stagnation point. The intake of matter at the centre via the inlet axis is thus reduced, being compensated by fluid flowing through low resistance corridors along the central vertical axis, above and below the central point. Furthermore, we propose and locally compute a modified Pakdel-McKinley criterion, thereby producing a scalar stability field and suggesting emergent peripheral stagnation points also indirectly contribute to the onset of time-dependent flow. (c) 2015 The Authors. Published by Elsevier B.V

A new viscoelastic benchmark flow: Stationary bifurcation in a cross-slot

AbstractIn this work we propose the cross-slot geometry as a candidate for a numerical benchmark flow problem for viscoelastic fluids. Extensive data of quantified accuracy is provided, obtained via Richardson extrapolation to the limit of infinite refinement using results for three different mesh resolutions, for the upper-convected Maxwell, Oldroyd-B and the linear form of the simplified Phan-Thien–Tanner constitutive models. Furthermore, we consider two types of flow geometry having either sharp or rounded corners, the latter with a radius of curvature equal to 5% of the channel’s width. We show that for all models the inertialess steady symmetric flow may undergo a bifurcation to a steady asymmetric configuration, followed by a second transition to time-dependent flow, which is in qualitative agreement with previous experimental observations for low Reynolds number flows. The critical Deborah number for both transitions is quantified and a set of standard parameters is proposed for benchmarking purposes

Variations in Inflammatory Markers in Acute Myocardial Infarction: a Longitudinal Study

Actualmente a inflamação é considerada uma componente importante na aterosclerose, desde o seu início até à ruptura da placa seguida de trombose e da progressiva obstrução do vaso. A ruptura da cápsula fibrótica da placa expõe factores de tecido presentes no seu núcleo necrótico que induzem o processo inflamatório, promovendo a adesão celular e a coagulação e que conduzem à formação do trombo. Por seu turno, várias citocinas e moléculas de adesão celular contribuem activamente para o desenvolvimento da placa. Em particular a citocina TNF-a e a molécula de adesão intercelular (ICAM-1) poderão ser indicadoras de inflamação enquanto que as formas solúveis de P-selectina e de CD40 ligando (sCD40L) poderão dar a magnitude da activação plaquetária. Neste trabalho foram estudados 17 doentes com enfarte de miocárdio submetidos a angioplastia (grupo AMI) e 16 doentes com confirmação angiográfica de ausência de doença coronária. Os doentes do grupo AMI foram seguidos nas primeiras 24h de evolução do enfarte agudo de miocárdio antes da administração de medicação e da intervenção angiográfica e ao longo do período de recuperação, 2 e 40 dias após enfarte. Foram medidas no soro por imunoensaio as concentrações de TNF-a e das formas solúveis de CD40L, ICAM-1 e P-selectina. Foram observadas variações significativas de sP-selectina relativamente aos controlos. Imediatamente após o enfarte de miocárdio verificou-se um aumento de sP-selectina, seguido de uma descida brusca dos seus níveis às 48h, e de um incremento para valores idênticos aos observados no grupo de controlo ao 40º dia. As variações observadas nas concentrações de sCD40L não foram significativas relativamente aos controlos. No entanto, verificou-se uma tendência de diminuição da concentração até 48h após o enfarte de miocárdio, seguindo-se um aumento que atingiu valores ligeiramente superiores ao do grupo controlo no 40º dia. As concentrações de TNF-a medidas foram sistematicamente superiores às verificadas no grupo controlo, tendo-se ainda observado uma subida gradual desde o enfarte de miocárdio até ao 40º dia, sendo este incremento significativo. Os valores de sICAM-1 não apresentaram quaisquer variações após o enfarte nem relativamente ao grupo controlo. As variações observadas sugerem um papel importante destes marcadores no processo inflamatório e na evolução do enfarte de miocárdio. O aumento brusco da concentração de sP-selectina após o enfarte de miocárdio evidencia a activação plaquetária e trombose. Na evolução do enfarte, e à medida que as variáveis hemodinâmicas retornam a valores estáveis, devido à medicação aplicada, o aumento de sCD40L e TNF-a em circulação pode reflectir o papel destas moléculas na recuperação endotelial e do miocárdio

Divergent trophic responses to biogeographic and environmental gradients

Following environmental changes, communities disassemble and reassemble in seemingly unpredictable ways. Whether species respond to such changes individualistically or collectively (e.g. as functional groups) is still unclear. To address this question, we used an extensive new dataset for the lake communities in the Azores' archipelago to test whether: 1) individual species respond concordantly within trophic groups; 2) trophic groups respond concordantly to biogeographic and environmental gradients. Spatial concordance in individual species distributions within trophic groups was always greater than expected by chance. In contrast, trophic groups varied non-concordantly along biogeographic and environmental gradients revealing idiosyncratic responses to them. Whether communities respond individualistically to environmental gradients thus depends on the functional resolution of the data. Our study challenges the view that modelling environmental change effects on biodiversity always requires an individualist approach. Instead, it finds support for the longstanding idea that communities might be modelled as a cohort if the functional resolution is appropriate

Ultrastructure of the Intramandibular Gland of Workers and Queens of the Stingless Bee, Melipona quadrifasciata

The intramandibular glands of workers and queens of Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae), at different ages and from different functional groups, were studied using light and transmission electron microscopy. The results demonstrated that these glands are composed of two types of secretory structures: 1.A hypertrophied epidermis on the dorsal side of the mandible that is an epithelial gland. 2. Free secretory cells filling the inner spaces of the appendices that constitute a unicellular gland. The epithelial gland is larger in the young (1-2-day-old workers), and the gland becomes involuted during the nurse worker stage. The unicellular glands of the workers posses some secretion during all of the studied phases, but secretory activity is more intensive in the foraging workers. Vesicles of secretion are absent in the unicellular glands of queens. These results demonstrate that these glands show functional adaptations in different castes corresponding to the functions of each caste

Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum

Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx's ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx's chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.We thank Frederico Silva for help with size-exclusion chromatography experiments, and Ana G. Gomes-Alves and Ricardo Silva for constructing the pSSU-PHLEO-infantum-MTS.His.THR-mTXNPx plasmid. This work was supported by National Institutes of Health Grant GM065318 (to U.J.) and Project "NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions" cofunded by Programa Operacional Regional do Norte under the Quadro de Referencia Estrategico Nacional, through Fundo Europeu de Desenvolvimento Regional, and by the Portuguese Foundation for Science and Technology (FCT) (A.M.T.). F.T. and H.C. were supported by Portuguese FCT Fellowships SFRH/BD/70438/2010 and SFRH/BPD/80836/2011, respectively