Papel de la serotonina periférica en la secreción de insulina y la homeostasis de la glucosa

La función más conocida de la serotonina (5-Hidroxi-triptamina, 5HT) se refiere a su acción en el Sistema Nervioso Central (SNC). Sin embargo, la mayoría de la 5HT corporal se genera periféricamente, principalmente en las células enterocromafines del intestino. Se ha descrito que la célula β-pancreática posee un sistema serotoninér-gico propio que le permite sintetizar, almacenar, secretar y responder a la 5HT extracelular a través de sus receptores, de los que se conocen numerosos subtipos agrupados en 7 familias (Htr1-7). Interesantemente, la 5HT se libera conjuntamente con la insulina y sólo recientemente se ha descifrado parte de su significado biológico, que incluiría una compleja combinación de efectos intra y extra-celulares que eventualmente podrían jugar un papel en la regulación de la secreción de esta hormona. De forma fisiológica, la expresión de las enzimas involucradas en la síntesis de 5HT y de sus receptores se modifica marcadamente en células β durante la gestación, en coincidencia con un incremento en el potencial secretor de insulina (vía la acción del receptor ionotrópico Htr3a) y un aumento en la masa de células β (vía la acción de receptores Htr1d y Htr2b). En otros tejidos, se ha sugerido que la 5HT procedente del intestino promueve la gluconeogéne-sis hepática y la lipólisis en adipocitos durante el ayuno, por medio de su acción sobre el receptor Htr2b. En conjunto, estos hallazgos sugieren que la 5HT periférica podría tener un rol importante en la homeostasis de la glucosa por medio de la expresión y activación diferencial de receptores de superficie en células clave, tales como hepatocitos, adipocitos y células β-pancreáticas.

I. Quantal effects in biochemical cooperativity and a proposed mechanism for the differentiation of calcium signaling in synaptic plasticity. II. Evolutionary algorithms for the optimization of methods in computational chemistry

In Part 1 of this thesis, we propose that biochemical cooperativity is a fundamentally non-ideal process. We show quantal effects underlying biochemical cooperativity and highlight apparent ergodic breaking at small volumes. The apparent ergodic breaking manifests itself in a divergence of deterministic and stochastic models. We further predict that this divergence of deterministic and stochastic results is a failure of the deterministic methods rather than an issue of stochastic simulations.

Ergodic breaking at small volumes may allow these molecular complexes to function as switches to a greater degree than has previously been shown. We propose that this ergodic breaking is a phenomenon that the synapse might exploit to differentiate Ca$^{2+}$ signaling that would lead to either the strengthening or weakening of a synapse. Techniques such as lattice-based statistics and rule-based modeling are tools that allow us to directly confront this non-ideality. A natural next step to understanding the chemical physics that underlies these processes is to consider \textit{in silico} specifically atomistic simulation methods that might augment our modeling efforts.

In the second part of this thesis, we use evolutionary algorithms to optimize \textit{in silico} methods that might be used to describe biochemical processes at the subcellular and molecular levels. While we have applied evolutionary algorithms to several methods, this thesis will focus on the optimization of charge equilibration methods. Accurate charges are essential to understanding the electrostatic interactions that are involved in ligand binding, as frequently discussed in the first part of this thesis.

Biochemical studies of postsynaptic density signaling proteins with a focus on synaptic GTPase activating protein and PDZ domains

Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP’s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP’s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.

The postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.

Homeostasis del hábitat. Articulación entre lo socio funcional y el medio natural

Bogotá (Colombia): Universidad de La Salle. Facultad de Ciencias del Hábitat. Programa de Arquitectura

Growing up in a bubble: using germ-free animals to assess the influence of the gut microbiota on brain and behavior

There is a growing recognition of the importance of the commensal intestinal microbiota in the development and later function of the central nervous system. Research using germ-free mice (mice raised without any exposure to microorganisms) has provided some of the most persuasive evidence for a role of these bacteria in gut-brain signalling. Key findings show that the microbiota is necessary for normal stress responsivity, anxiety-like behaviors, sociability, and cognition. Furthermore, the microbiota maintains central nervous system homeostasis by regulating immune function and blood brain barrier integrity. Studies have also found that the gut microbiota influences neurotransmitter, synaptic, and neurotrophic signalling systems and neurogenesis. The principle advantage of the germ-free mouse model is in proof-of-principle studies and that a complete microbiota or defined consortiums of bacteria can be introduced at various developmental time points. However, a germ-free upbringing can induce permanent neurodevelopmental deficits that may deem the model unsuitable for specific scientific queries that do not involve early-life microbial deficiency. As such, alternatives and complementary strategies to the germ-free model are warranted and include antibiotic treatment to create microbiota-deficient animals at distinct time points across the lifespan. Increasing our understanding of the impact of the gut microbiota on brain and behavior has the potential to inform novel management strategies for stress-related gastrointestinal and neuropsychiatric disorders.

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG. Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

S-nitrosylation homeostasis in plants: key enzymes and regulatory pathways

The nitrosylated form of glutathione (GSNO) has been acknowledged to be the most important nitrosylating agent of the plant cell, and the tuning of its intracellular concentration is of pivotal importance for photosynthetic life. During my time as a PhD student, I focused my attention on the enzymatic systems involved in the degradation of GSNO. Hence, we decided to study the structural and catalytic features of alcohol dehydrogenases (GSNOR and ADH1) from the model land plant Arabidopsis thaliana (At). These enzymes displayed a very similar 3D structure except for their active site which might explain the extreme catalytic specialization of the two enzymes. They share NAD(H) as a cofactor, but only AtGSNOR was able to catalyze the reduction of GSNO whilst being ineffective in oxidizing ethanol. Moreover, our study on the enzyme from the unicellular green alga Chlamydomonas reinhardtii (Cr) revealed how this S-nitrosoglutathione reductase (GSNOR) specifically use NADH to catalyze GSNO reduction and how its activity responds to thiol-based post-translational modifications. Contextually, the presence of NADPH-dependent GSNO-degrading systems in algal protein extract was highlighted and resulted to be relatively efficient in this model organism. This activity could be ascribed to several proteins whose contribution has not been defined yet. Intriguingly, protein extract from GSNOR null mutants of Arabidopsis displayed an increased NADPH-dependent ability to degrade GSNO and our quantitative proteome profiling on the gsnor mutant revealed the overexpression of two class 4 aldo-keto reductases (AKR), specifically AtAKR4C8 and AtAKR4C9. Later, all four class 4 AKRs showed to possess a NADPH-dependent GSNO-degrading activity. Finally, we initiated a preliminary analysis to determine the kinetic parameters of several plant proteins, including GSNOR, AKR4Cs, and thioredoxins. These data suggested GSNOR to be the most effective enzyme in catalyzing GSNO reduction because of its extremely high catalytic proficiency compared to NADPH-dependent systems.

Obesity And Inflammation And The Effect On The Hematopoietic System.

Bone marrow is organized in specialized microenvironments known as 'marrow niches'. These are important for the maintenance of stem cells and their hematopoietic progenitors whose homeostasis also depends on other cell types present in the tissue. Extrinsic factors, such as infection and inflammatory states, may affect this system by causing cytokine dysregulation (imbalance in cytokine production) and changes in cell proliferation and self-renewal rates, and may also induce changes in the metabolism and cell cycle. Known to relate to chronic inflammation, obesity is responsible for systemic changes that are best studied in the cardiovascular system. Little is known regarding the changes in the hematopoietic system induced by the inflammatory state carried by obesity or the cell and molecular mechanisms involved. The understanding of the biological behavior of hematopoietic stem cells under obesity-induced chronic inflammation could help elucidate the pathophysiological mechanisms involved in other inflammatory processes, such as neoplastic diseases and bone marrow failure syndromes.

The Crosslinking Degree Controls The Mechanical, Rheological, And Swelling Properties Of Hyaluronic Acid Microparticles.

Viscosupplements, used for treating joint and cartilage diseases, restore the rheological properties of synovial fluid, regulate joint homeostasis and act as scaffolds for cell growth and tissue regeneration. Most viscosupplements are hydrogels composed of hyaluronic acid (HA) microparticles suspended in fluid HA. These microparticles are crosslinked with chemicals to assure their stability against enzyme degradation and to prolong the action of the viscosupplement. However, the crosslinking also modifies the mechanical, swelling and rheological properties of the HA microparticle hydrogels, with consequences on the effectiveness of the application. The aim of this study is to correlate the crosslinking degree (CD) with these properties to achieve modulation of HA/DVS microparticles through CD control. Because divinyl sulfone (DVS) is the usual crosslinker of HA in viscosupplements, we examined the effects of CD by preparing HA microparticles at 1:1, 2:1, 3:1, and 5:1 HA/DVS mass ratios. The CD was calculated from inductively coupled plasma spectrometry data. HA microparticles were previously sized to a mean diameter of 87.5 µm. Higher CD increased the viscoelasticity and the extrusion force and reduced the swelling of the HA microparticle hydrogels, which also showed Newtonian pseudoplastic behavior and were classified as covalent weak. The hydrogels were not cytotoxic to fibroblasts according to an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.

Impaired Incretin Secretion And Pancreatic Dysfunction With Older Age And Diabetes.

To estimate the impact of aging and diabetes on insulin sensitivity, beta-cell function, adipocytokines, and incretin production. Hyperglycemic clamps, arginine tests and meal tolerance tests were performed in 50 non-obese subjects to measure insulin sensitivity (IS) and insulin secretion as well as plasma levels of glucagon, GLP-1 and GIP. Patients with diabetes and healthy control subjects were divided into the following groups: middle-aged type 2 diabetes (MA-DM), aged Type 2 diabetes (A-DM) and middle-aged or aged subjects with normal glucose tolerance (MA-NGT or A-NGT). IS, as determined by the homeostasis model assessment, glucose infusion rate, and oral glucose insulin sensitivity, was reduced in the aged and DM groups compared with MA-NGT, but it was similar in the MA-DM and A-DM groups. Insulinogenic index, first and second phase insulin secretion and the disposition indices, but not insulin response to arginine, were reduced in the aged and DM groups. Postprandial glucagon production was higher in MA-DM compared to MA-NGT. Whereas the GLP-1 production was reduced in A-DM, no differences between groups were observed in GIP production. In non-obese subjects, diabetes and aging impair insulin sensitivity. Insulin production is reduced by aging, and diabetes exacerbates this condition. Aging associated defects superimposed diabetic physiopathology, particularly regarding GLP-1 production. On the other hand, the glucose-independent secretion of insulin was preserved. Knowledge of the complex relationship between aging and diabetes could support the development of physiopathological and pharmacological based therapies.

Overexpression Of Ucp1 In Tobacco Induces Mitochondrial Biogenesis And Amplifies A Broad Stress Response.

Uncoupling protein one (UCP1) is a mitochondrial inner membrane protein capable of uncoupling the electrochemical gradient from adenosine-5'-triphosphate (ATP) synthesis, dissipating energy as heat. UCP1 plays a central role in nonshivering thermogenesis in the brown adipose tissue (BAT) of hibernating animals and small rodents. A UCP1 ortholog also occurs in plants, and aside from its role in uncoupling respiration from ATP synthesis, thereby wasting energy, it plays a beneficial role in the plant response to several abiotic stresses, possibly by decreasing the production of reactive oxygen species (ROS) and regulating cellular redox homeostasis. However, the molecular mechanisms by which UCP1 is associated with stress tolerance remain unknown. Here, we report that the overexpression of UCP1 increases mitochondrial biogenesis, increases the uncoupled respiration of isolated mitochondria, and decreases cellular ATP concentration. We observed that the overexpression of UCP1 alters mitochondrial bioenergetics and modulates mitochondrial-nuclear communication, inducing the upregulation of hundreds of nuclear- and mitochondrial-encoded mitochondrial proteins. Electron microscopy analysis showed that these metabolic changes were associated with alterations in mitochondrial number, area and morphology. Surprisingly, UCP1 overexpression also induces the upregulation of hundreds of stress-responsive genes, including some involved in the antioxidant defense system, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). As a consequence of the increased UCP1 activity and increased expression of oxidative stress-responsive genes, the UCP1-overexpressing plants showed reduced ROS accumulation. These beneficial metabolic effects may be responsible for the better performance of UCP1-overexpressing lines in low pH, high salt, high osmolarity, low temperature, and oxidative stress conditions. Overexpression of UCP1 in the mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions.

Influence Of Gastric Emptying On The Control Of Postprandial Glycemia: Physiology And Therapeutic Implications.

The maintenance of glucose homeostasis is complex and involves, besides the secretion and action of insulin and glucagon, a hormonal and neural mechanism, regulating the rate of gastric emptying. This mechanism depends on extrinsic and intrinsic factors. Glucagon-like peptide-1 secretion regulates the speed of gastric emptying, contributing to the control of postprandial glycemia. The pharmacodynamic characteristics of various agents of this class can explain the effects more relevant in fasting or postprandial glucose, and can thus guide the individualized treatment, according to the clinical and pathophysiological features of each patient.

Magnesium-deficient High-fat Diet: Effects On Adiposity, Lipid Profile And Insulin Sensitivity In Growing Rats.

To determine if magnesium deficiency aggravates the effects of a high-fat diet in growing rats in terms of obesity, lipid profile and insulin resistance. The study population comprised 48 newly weaned male Wistar Hannover rats distributed into four groups according to diet, namely, control group (CT; n = 8), control diet provided ad libitum; pair-feeding control group (PF; n = 16), control diet but in the same controlled amount as animals that received high-fat diets; high-fat diet group (HF; n = 12), and magnesium-deficient high-fat diet group (HFMg(-); n = 12). The parameters investigated were adiposity index, lipid profile, magnesium status, insulin sensitivity and the phosphorylation of proteins involved in the insulin-signaling pathway, i.e. insulin receptor β-subunit, insulin receptor substrate 1 and protein kinase B. The HF and HFMg(-) groups were similar regarding gain in body mass, adiposity index and lipid profile, but were significantly different from the PF group. The HFMg(-) group exhibited alterations in magnesium homeostasis as revealed by the reduction in urinary and bone concentrations of the mineral. No inter-group differences were observed regarding glucose homeostasis. Protein phosphorylation in the insulin-signaling pathway was significantly reduced in the high-fat groups compared with the control groups, demonstrating that the intake of fat-rich diets increased insulin resistance, a syndrome that was aggravated by magnesium deficiency. Under the experimental conditions tested, the intake of a magnesium-deficient high-fat diet led to alterations in the insulin-signaling pathway and, consequently, increased insulin resistance.

Neuromuscular Activity Of Bothrops Fonsecai Snake Venom In Vertebrate Preparations.

The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160μg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160μg/ml, respectively) and attenuated the contractures to 110μM ACh (78-100% inhibition) and 40mM KCl (45-90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200μg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200μg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K(+) were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom.