The subcellular fractionation properties and function of insulin receptor substrate-1 (IRS-1) are independent of cytoskeletal integrity

Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, individually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim-/- cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton. (c) 2006 Elsevier Ltd. All rights reserved.

Investigation of potential intervention targets to improve insulin action: a therapeutic approach

Impaired insulin action (insulin resistance) is a key factor in the pathogenesis of diabetes mellitus. To investigate therapeutic targets against insulin resistance, this thesis explores the mechanism of action of pharmacological agents and exogenous peptides known or suspected to modify insulin action. These included leptin, a hormone primarily involved in the regulation of body weight; sibutramine, an antiobesity agent; plant-derived compounds (pinitol and chamaemeloside) and agents known to affect insulin sensitivity, e.g. metformin, tolbutamide, thiazolidinediones, vanadyl sulphate and thioctic acid. Models used for investigation included the L6 skeletal muscle cell line and isolated skeletal muscles. In vivo studies were undertaken to investigate glycaemia, insulinaemia, satiety and body weight in streptozotocin-induced diabetic mice and obese (ob/ob) mice. Leptin acutely altered insulin action in skeletal muscle cells via the short form of the leptin receptor. This direct action of leptin was mediated via a pathway involving PI 3-kinase but not Jak2. The active metabolites of sibutramine had antidiabetic properties in vivo and directly improved insulin sensitivity in vitro. This effect appeared to be conducted via a non-PI 3-kinase-mediated increase in protein synthesis with facilitated glucose transport, and was independent of the serotonin and noradrenaline reuptake inhibition produced by sibutramine. Pinitol (a methyl inositol) had an insulin mimetic effect and was an effective glucose-lowering agent in insulinopenic states, acting directly on skeletal muscle. Conversely chamaemeloside appeared to improve glucose tolerance without directly altering glucose transport. Metformin directly increased basal glucose uptake independently of PI 3-kinase, possibly via an increase in the intrinsic activity of glucose transporters. Neither tolbutamide nor thiazolidinediones directly altered insulin sensitivity in L6 skeletal muscle cells: however vanadyl sulphate and thioctic acid increased glucose transport but appeared to exert toxic effects at therapeutic concentrations. Examination of glucose transport in skeletal muscle in this thesis has identified various components of post-receptor insulin signalling pathways which may be targeted to ameliorate insulin resistance. Type 2 Diabetes Mellitus Obesity L6 Skeletal Muscle Cells Glucose Transport Insulin Signalling 2

Role of β-adrenergic receptors in the anti-obesity and anti-diabetic effects of zinc-α2-glycoprotien (ZAG)

Objectives: The goal of the current study is to determine whether the ß-adrenoreceptor (ß-AR) plays a role in the anti-obesity and anti-diabetic effects of zinc-a2-glycoprotein (ZAG). Material and methods: This has been investigated in CHO-K1 cells transfected with the human ß1-, ß2-, ß3-AR and in ob/ob mice. Cyclic AMP assays were carried out along with binding studies. Ob/ob mice were treated with ZAG and glucose transportation and insulin were examined in the presence or absence of propranolol. Results: ZAG bound to the ß3-AR with higher affinity (Kd 46±1nM) than the ß2-AR (Kd 71±3nM) while there was no binding to the ß1-AR, and this correlated with the increases in cyclic AMP in CHO-K1 cells transfected with the various ß-AR and treated with ZAG. Treatment of ob/ob mice with ZAG increased protein expression of ß3-AR in gastrocnemius muscle, and in white and brown adipose tissues, but had no effect on expression of ß1- and ß2-AR. A reduction of body weight was seen and urinary glucose excretion, increase in body temperature, reduction in maximal plasma glucose and insulin levels in the oral glucose tolerance test, and stimulation of glucose transport into skeletal muscle and adipose tissue, were completely attenuated by the non-specific ß-AR antagonist propranolol. Conclusion: The results suggest that the effects of ZAG on body weight and insulin sensitivity in ob/ob mice are manifested through a ß-3AR, or possibly a ß2-AR.

Movement Effects on the Flow Physics and Nutrient Delivery in Engineered Valvular Tissues

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this study, we computationally quantified the i) magnitude of fluid-induced shear stresses; ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter, and iii) glucose and oxygen mass transport profiles. Noting that sample cyclic flexure induces a high degree of oscillatory shear stress (OSS), we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: 1) no flow, no flexure (control group), 2) steady flow-alone, 3) cyclic flexure-alone and 4) combined steady flow and cyclic flexure environments. We also coupled a diffusion and convention mass transport equation to the simulated system. We found that the coexistence of both OSS and appreciable shear stress magnitudes, described by the newly introduced parameter OSI-t , explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress. In addition, our simulations showed that mass transport of glucose and oxygen is enhanced by sample movement at low sample porosities, but did not play a role in highly porous scaffolds. Preliminary in-house in vitro experiments showed that cell proliferation and phenotype is enhanced in OSI-t environments.

Concussion: Examining the Effect of Neuronal Oxidative Stress on the Pathophysiology of Brain and Blood Brain Barrier Cells

Neuronal stretching during concussion alters glucose transport and reduces neuronal viability, also affecting other cells in the brain and the Blood Brain Barrier (BBB). Our hypothesis is that oxidative stress (OS) generated in neurons during concussions contributes to this outcome. To validate this, we investigated: (1) whether OS independently causes alterations in brain and BBB cells, namely human neuron-like, neuroblastoma cells (NCs), astrocyte cells (ACs) and brain microvascular endothelial cells (ECs), and (2) whether OS originated in NCs (as in concussion) is responsible for causing the subsequent alterations observed in ACs and ECs. We used H2O2 treatment to mimic OS, validated by examining the resulting reactive oxygen species, and evaluated alterations in cell morphology, expression and localization of the glucose transporter GLUT1, and the overall cell viability. Our results showed that OS, either directly affecting each cell type or originally affecting NCs, caused changes in several morphological parameters (surface area, Feret diameter, circularity, inter-cellular distance), slightly varied GLUT1 expression and lowered the overall cell viability of all NCs, ACs, and ECs. Therefore, we can conclude that oxidative stress, which is known to be generated during concussion, caused alterations in NCs, ACs, and ECs whether independently originated in each cell or when originated in the NCs and could further propagate the ACs and ECs.

Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells

Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.

La résistance à l’insuline en insuffisance rénale chronique et le risque de développer un diabète de type 2 : un cercle vicieux

L’insuffisance rénale chronique (IRC) est caractérisée par de multiples déséquilibres homéostatiques tels que la résistance à l’insuline. Peu d’études se sont intéressées aux mécanismes sous-jacents à cette résistance à l’insuline en IRC. De plus, il est méconnu si cette résistance à l’insuline peut mener au développement d’un diabète de type II chez des patients prédisposés. Dans un modèle d’IRC, le rat Sprague-Dawley (CD) néphrectomisé 5/6e, on observe une corrélation entre la gravité de l’atteinte rénale, évaluée par la créatinine sérique, et l’hyperglycémie, évaluée par la fructosamine sérique (R2 = 0.6982, p < 0.0001). Cependant, cet état hyperglycémique n’est pas observable lors d’une glycémie à jeun. Lors d’un test de tolérance au glucose, on observe une plus grande élévation de la glycémie (AUC 1.25 fois, p < 0.0001) chez le rat atteint d’IRC. Par contre, la sécrétion d’insuline au cours de ce même test n’augmente pas significativement (AUC ≈ 1.30 fois, N.S.) en comparaison aux rats témoins. Malgré une élévation des taux d’insuline en IRC suivant un bolus de glucose, les tissus périphériques ne montrent pas d’augmentation de la captation du glucose sanguin suggérant un défaut d’expression et/ou de fonction des transporteurs de glucose chez ces rats. En effet, on observe une diminution de ces transporteurs dans divers tissus impliqués dans le métabolisme du glucose tel que le foie (≈ 0.60 fois, p < 0.01) et le muscle (GLUT1 0.73 fois, p < 0.05; GLUT4 0.69 fois, p < 0.01). En conséquence, une diminution significative du transport insulinodépendant du glucose est observable dans le muscle des rats atteint d’IRC (≈ 0.63 fois, p < 0.0001). Puisque les muscles sont responsables de la majorité de la captation insulinodépendante du glucose, la diminution de l’expression du GLUT4 pourrait être associée à la résistance à l’insuline observée en IRC. La modulation de l’expression des transporteurs de glucose pourrait être à l’origine de la résistance à l’insuline en IRC. Cela dit, d’autres mécanismes peuvent aussi être impliqués. En dépit de cette importante perturbation du transport du glucose, nous n’avons pas observé de cas de diabète de type II chez le rat CD atteint d’IRC. Dans un modèle de rat atteint d’un syndrome métabolique, le rat Zucker Leprfa/fa, l’IRC provoque une forte hyperglycémie à jeun (1.5 fois, p < 0.0001). De plus, l’IRC chez le rat Zucker provoque une réponse glycémique (AUC 1.80 fois, p < 0.0001) exagérée lors d’un test de tolérance au glucose. Une forte résistance à l’insuline est mesurée au niveau des muscles puisque la dose usuelle d’insuline (2mU/mL) n’est pas suffisante pour stimuler la captation du glucose chez le rat Zucker atteint d’IRC. De plus, une modulation similaire des transporteurs de glucose peut être observée chez ces deux espèces. Par contre, environ 30% (p < 0.001) des rats Zucker atteints d’IRC avaient une glycosurie. L’IRC en soi ne mènerait donc pas au développement d’un diabète de type II. Par contre, lorsqu’une résistance à l’insuline est présente antérieurement au développement d’une IRC, cela pourrait précipiter l’apparition d’un diabète de type II chez ces patients prédisposés.

Movement effects on the flow physics and nutrient delivery in engineered valvular tissues

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this study, we computationally quantified the i) magnitude of fluid-induced shear stresses; ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter, and iii) glucose and oxygen mass transport profiles. Noting that sample cyclic flexure induces a high degree of oscillatory shear stress (OSS), we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: 1) no flow, no flexure (control group), 2) steady flow-alone, 3) cyclic flexure-alone and 4) combined steady flow and cyclic flexure environments. We also coupled a diffusion and convention mass transport equation to the simulated system. We found that the coexistence of both OSS and appreciable shear stress magnitudes, described by the newly introduced parameter OSI-:τ: explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress. In addition, our simulations showed that mass transport of glucose and oxygen is enhanced by sample movement at low sample porosities, but did not play a role in highly porous scaffolds. Preliminary in-house in vitro experiments showed that cell proliferation and phenotype is enhanced in OSI-:τ: environments.^

La résistance à l’insuline en insuffisance rénale chronique et le risque de développer un diabète de type 2 : un cercle vicieux

L’insuffisance rénale chronique (IRC) est caractérisée par de multiples déséquilibres homéostatiques tels que la résistance à l’insuline. Peu d’études se sont intéressées aux mécanismes sous-jacents à cette résistance à l’insuline en IRC. De plus, il est méconnu si cette résistance à l’insuline peut mener au développement d’un diabète de type II chez des patients prédisposés. Dans un modèle d’IRC, le rat Sprague-Dawley (CD) néphrectomisé 5/6e, on observe une corrélation entre la gravité de l’atteinte rénale, évaluée par la créatinine sérique, et l’hyperglycémie, évaluée par la fructosamine sérique (R2 = 0.6982, p < 0.0001). Cependant, cet état hyperglycémique n’est pas observable lors d’une glycémie à jeun. Lors d’un test de tolérance au glucose, on observe une plus grande élévation de la glycémie (AUC 1.25 fois, p < 0.0001) chez le rat atteint d’IRC. Par contre, la sécrétion d’insuline au cours de ce même test n’augmente pas significativement (AUC ≈ 1.30 fois, N.S.) en comparaison aux rats témoins. Malgré une élévation des taux d’insuline en IRC suivant un bolus de glucose, les tissus périphériques ne montrent pas d’augmentation de la captation du glucose sanguin suggérant un défaut d’expression et/ou de fonction des transporteurs de glucose chez ces rats. En effet, on observe une diminution de ces transporteurs dans divers tissus impliqués dans le métabolisme du glucose tel que le foie (≈ 0.60 fois, p < 0.01) et le muscle (GLUT1 0.73 fois, p < 0.05; GLUT4 0.69 fois, p < 0.01). En conséquence, une diminution significative du transport insulinodépendant du glucose est observable dans le muscle des rats atteint d’IRC (≈ 0.63 fois, p < 0.0001). Puisque les muscles sont responsables de la majorité de la captation insulinodépendante du glucose, la diminution de l’expression du GLUT4 pourrait être associée à la résistance à l’insuline observée en IRC. La modulation de l’expression des transporteurs de glucose pourrait être à l’origine de la résistance à l’insuline en IRC. Cela dit, d’autres mécanismes peuvent aussi être impliqués. En dépit de cette importante perturbation du transport du glucose, nous n’avons pas observé de cas de diabète de type II chez le rat CD atteint d’IRC. Dans un modèle de rat atteint d’un syndrome métabolique, le rat Zucker Leprfa/fa, l’IRC provoque une forte hyperglycémie à jeun (1.5 fois, p < 0.0001). De plus, l’IRC chez le rat Zucker provoque une réponse glycémique (AUC 1.80 fois, p < 0.0001) exagérée lors d’un test de tolérance au glucose. Une forte résistance à l’insuline est mesurée au niveau des muscles puisque la dose usuelle d’insuline (2mU/mL) n’est pas suffisante pour stimuler la captation du glucose chez le rat Zucker atteint d’IRC. De plus, une modulation similaire des transporteurs de glucose peut être observée chez ces deux espèces. Par contre, environ 30% (p < 0.001) des rats Zucker atteints d’IRC avaient une glycosurie. L’IRC en soi ne mènerait donc pas au développement d’un diabète de type II. Par contre, lorsqu’une résistance à l’insuline est présente antérieurement au développement d’une IRC, cela pourrait précipiter l’apparition d’un diabète de type II chez ces patients prédisposés.

Regulation of intestinal cholesterol transport and metabolism by high glucose levels = Régulation intestinale du transport et du métabolisme du cholestérol par le glucose

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Transepithelial transport of glucose and mRNA of glucose transporters in the small intestine of rats with iron-deficiency anemia

Objective: To evaluate the transepithelial transport of sodium, glucose, potassium, and water and the mRNA level of the sodium-glucose cotransporter (SGLT1) and the facilitated sugar transporter (GLUT2) in the small intestine of iron-deficient rats. Methods: After 6 wk of receiving diets with low or normal iron content, rats (Wistar-EPM) were subjected to two experiments: 1) evaluation of the transepithelial transport of sodium, glucose, potassium, and water by an ""in vivo"" experimental model of intestinal perfusion and 2) determination of relative SGLT1 and GLUT2 mRNA levels in the proximal, intermediate, and distal portions of the small intestine by the northern blotting technique. Results: Hemoglobin and hepatic iron levels were statistically lower in the anemic rats. The mean transepithelial transports of sodium (-33.0 mu Eq . min(-1) . cm(-1)), glucose (426.0 mu M . min(-1) . cm(-1)), and water (0.4 mu L . min(-1) . cm(-1)) in the small intestine of the anemic rats were significantly lower than in the control group (349.1 mu Eq . min(-1) cm(-1), 842.6 mu M . min(-1) . cm(-1), and 4.3 mu l . min(-1) cm(-1), respectively, P < 0.05). The transepithelial transport of potassium was similar for both groups. The relative SGLT1 mRNA levels of the anemic rats in the intermediate (1.796 +/- 0.659 AU) and distal (1.901 +/- 0.766 AU) segments were significantly higher than the values for the control rats (intermediate 1.262 +/- 0.450 AU, distal 1.244 +/- 0.407 AU). No significant difference was observed for the relative SLGT1 mRNA levels in the proximal segment or for the GLUT2 mRNA levels in all segments. Conclusion: Iron deficiency decreases the absorption of glucose, sodium, and water and increases SGLT1 mRNA in the intermediate and distal segments of the small intestine of rats. (C) 2011 Elsevier Inc. All rights reserved.

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9)

Placental Uric Acid Transport System: Glucose Transporter 9 (SLC2A9). INTRODUCTION: Pre-eclampsia, a pregnancy-specific disease, contributes substantially to perinatal morbidity and mortality of both the mother and her child. Pre-eclampsia is often associated with high maternal urate serum levels, which in turn has been shown to play a role in the pathogenesis of this disease. The aim of this study was to investigate the glucose transporter GLUT9-mediated placental uric acid transport system. METHODS: In this study western blot, immunofluorescence techniques as well as a transepithelial transport (Transwell) model were used to assess GLUT9 protein expression and, respectively, uric acid transport activity. Electrophysiological techniques and transmission electron microscopy (TEM) were used to characterize the properties and the structure of GLUT9. RESULTS: Uric acid is transported across a BeWo choriocarcinoma cell monolayer with 530 pmol/min. We could successfully overexpress and for the first time purify the GLUT9b isoform using the Xenopus laevis oocytes expression system. Chloride seems to modulate the urate transport system. TEM revealed that GLUT9b isoform is present as monomer and dimmer in the Xenopus laevis overexpression model. A class average of all the particles allowed us to develop a first model of human GLUT9b structure, which was derived from the published crystal structure of the bacterial homologue of GLUT1-4. CONCLUSIONS: In vitro the “materno-fetal” transport of uric acid is slow indicating that in vivo the fetus might be protected from short-term fluctuations of maternal urate serum levels. The low-resolution structure obtained from TEM validates the proposed homology model regarding the structure of human GLUT9b. In ongoing studies this model is used to perform virtual screening to identify novel modulators of the urate transport system enabling the development of novel therapies in pregnancy complications.

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite1

An NADPH-dependent NO2−-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP+ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO2− reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO2− reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP+. NADP+ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO2− reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO2− reduction and (b) consume NADP+, releasing FNR from NADP+ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO2− reduction observed in vitro is capable of accounting for the observed rates of dark NO3− assimilation by C. reinhardtii.

Transport d'eau généré par le cotransport Na+/glucose : nouvelle interprétation basée sur les effets distincts des flux entrants de cations et de glucose

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Régulation du transport de glucose par le [bêta]-hydroxybutyrate dans le muscle cardiaque

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.