The transport of glucose across the plasma membrane is a fundamental mechanism to provide cells with its basic requirements for energy yielding processes. It is also vital for clearing glucose from blood into tissues, a process normally stimulated by the hormone insulin in mammals. The sympathetic nervous system, normally activated during stress, also regulates glucose transport. The sympathetic neurotransmitter noradrenaline, acts on the family of adrenergic receptors (ARs). An important subtype of the AR family is the β-AR, which is subdivided into the β1, β2, and β3-AR. Glucose is transported across the plasma membrane by the family of glucose transporters (GLUT1-12, and HMIT). In this thesis, I have investigated the β-AR regulation of GLUT1 and 4, and glucose uptake, in skeletal muscle cells and brown adipocytes in culture, model systems which correspond to metabolically active, sympathetically innervated and insulin-sensitive tissues. In brown adipocytes, activation of the β3-ARs induced the expression of GLUT1, resulting in a large increase of glucose uptake. In skeletal myotubes, we postulate there is a possible mechanism where β2-ARs can regulate the intrinsic activity of GLUT1. We found that insulin signaling, but not β-adrenergic signaling, mediated glucose uptake through class I phosphatidylinositol 3-kinase (PI3K). The β-adrenergic signaling to glucose uptake appeared to involve a PI3K related kinase (PIKK), in both skeletal myotubes and brown adipocytes. Furthermore, the increase of glucose uptake by β-ARs in brown adipocytes is partially mediated by AMP-activated protein kinase (AMPK). However, in an artificially constructed system, with cells expressing GLUT4 and β2-ARs, both insulin and β-adrenergic activation translocated GLUT4 and increased glucose uptake. These results show that β-adrenergic signaling increase glucose uptake by regulating glucose transporters through distinct pathways, in skeletal myotubes and brown adipocytes.

No description available.

Obesity and Diabetes, the major cause for morbidity and mortality in United States raises a general curiosity regarding health care expenses when talked about treating them. Every year approximately 300,000 US adults die of reasons associated to obesity and diabetes, becoming the sixth leading cause of death. The prevalence of those diagnosed with diabetes witnessed an exponential curve in the last decade and for the year 2011 about 8.3% of the population in the US has been diagnosed with diabetes and it is predicted that in the year 2030 the prevalence of diabetes is to reach 4.4% globally. Type 2 diabetes is a condition, which develops when the body no longer makes enough insulin or when the insulin so produced does not work effectively. In reaction to the increase in obesity, treatments for obesity became more common especially the pharmacological treatments. Since this treatment also required one to change their lifestyle and food habits, bariatric surgeries were considered as an option to treat obesity and diabetes. A range of surgical procedures have been used to stimulate weight loss for obese patients. These procedures resulted in weight loss by restricting the size of the stomach (Gastric Banding) or bypassing a portion of the intestine (Gastric Bypass). Roux-en-Y Gastric Bypass (RYGB) accomplishes weight loss during a combination of gastric restriction and malabsorption. Reduction of the stomach to a small gastric pouch results in feelings of satiety. The RYGB procedure has been performed regularly since the early 1980s; it was first performed laparoscopically in the early 1990s. Ileal interposition (IT) is a surgical procedure where a section of ileum is snipped and moved closer to the jejunum. It is said that the food takes just ten minutes to reach the ileum instead of an hour after this procedure. The ileum produces Glucagon like Peptide-1 (GLP-1) which helps in insulin secretion. Glucose is a key stimulator for mammals and is derived from the diet consumed, transferred from the circulation into the target cells. Glucose penetrates the eukaryotic cells through membrane associated carrier proteins, the Na+ coupled glucose transporter (SGLT-1) and the glucose transporter (GLUT). These transporters are structurally and functionally distinct. The main research question was "are the receptors involved in glucose transport across the membrane (GLUT2 and SGLT1) important for Ileal Interposition"? With experiments like real time PCR (qPCR) and immunohistochemistry (IHC), we have observed the differences in the expression of these receptors with respect to the location and organ. Ileal interposition showed a significant difference (p<0.01) compared to sham-operated rats in the expression of GLUT2 in the gluconeogenic organs. The increased GLUT2 levels in ileal interposition may explain glucose sensitivity and these data emphasize the need for GLUT2 to maintain a positive glucose homeostasis and further study on SGLT1/GLUT2 influence on gluconeogenesis.

Previous epidemiological studies have shown that the diabetic population has a poor oral status compared to healthy subjects. While changes of the local microcirculation observed during diabetes can somehow explain periodontal pathologies this hypothesis does not stand for the carious decay. We propose the hypothesis that the increase of carious incidence might be linked to changes in the salivary composition, such as an increase of salivary glucose concentrations.The amount of glucose in saliva has been widely discussed in various scientific articles. Subsequently, we performed an initial study that allowed us to confirm that the concentration and excretion of glucose in the saliva were higher in diabetic patients compared to normal patients. In diabetic patients, the relative increase in salivary glucose concentration is even comparable to the rise of blood glucose.We conducted experimental studies in animals in order to investigate the presence and the level of expression of glucose transporters (GLUT1, GLUT2, GLUT4, SGLT1) in rat parotid glands. Through immunohistochemical labeling techniques, gene expression studies and protein expression studies, we demonstrated that GLUT1, GLUT4 and SGLT1 were found in rat parotid glands of normal and diabetic rats. Functional studies targeting the activity and the flux through those glucose transporters strengthened our hypothesis on the potential role of glucose transporter in salivary glucose.This original work introduce for the first time the hypothesis of a secretion mechanism of glucose by the salivary glands through GLUT1 & SGLT1 transporters, the first one being located on the apical and baso-lateral membranes of acinar cells, while the second one is located on the baso-lateral membrane. Secretion of glucose by the parotid gland is no longer at any doubt even though further studies must now specify the specific mechanisms. / Doctorat en Sciences dentaires / info:eu-repo/semantics/nonPublished

Type 1 diabetes (T1D) results from a loss of functional insulin-producing pancreatic beta cells. The etiology of T1D is poorly understood, but the detection of infiltrating inflammatory cells in the pancreas and circulating autoantibodies has led to the common notion that an autoimmune process plays a central role in the pathogenesis of the disease. The aim of this doctoral thesis was to assess various aspects of the immunopathology of type 1 diabetes. To this purpose, studies have been conducted on pancreatic material from the Network for Pancreatic Organ Donors with Diabetes (nPOD) collection, the Nordic Network for Islet Transplantation, and the Diabetes Virus Detection (DiViD) study. Paper I is a study on pancreatic tissue from organ donors with varying duration of T1D as well as non-diabetic donors and subjects with other types of diabetes, in which persistent expression of glucose transporters was shown on the beta cell membrane despite several years of T1D. Glucose transporter 1 was also confirmed as the predominant glucose transporter on human pancreatic islets. In paper II, we report on signs of inflammation in the exocrine but not in the endocrine pancreas in non-diabetic organ donors with diabetes-related autoantibodies, suggesting that diabetes-associated autoantibodies can occur in response to unspecific pancreatic lesions. Paper III aimed to characterize the T cell-infiltration of pancreatic islets in material from recent-onset T1D patients. Insulitis was shown in all subjects, but with distinct differences in expression analysis of T- and B cell activation to cell-mediated allorejected kidney transplant. Also Paper IV was conducted on material from recent-onset cases and showed increased islet glucagon content, in combination with a reduced number of islets but sustained mean islet size. Together, these results provide expansion of our knowledge of the immunopathology in T1D, and will hopefully assist in bringing us towards a deeper understanding of T1D aetiology and eventually an effective cure.

The protein-energy malnutrition is one of the most serious nutritional problems that occur in developing countries. The energy and nutritional deficiency in early life can cause significant changes in different stages of development of central nervous system, since the cases of hyperplasia, hypertrophy and myelinization occur in more intense. The brain uses glucose as its main energy substrate. However, under certain physiological conditions, as during the weaning or during prolonged fasting, the cells may use other substrates in order to meet their metabolic needs. The transport of glucose to the nerve cells requires the presence of specific transporter proteins called GLUT s (Glucose Transporters) to perform the transport of glucose by facilitated diffusion. Besides the transporters of glucose, transporter proteins of monocarboxilatos (MCTs) are responsible for neuronal influx of lactate, MCT1 being the main responsible for providing energy to neurons during maturation of nervous tissue. The acetylcholine is a major neurotransmitter of the nervous system and is associated with the maintenance processes of attention, learning and memory. In the synaptic cleft acetylcholine is degraded by the enzyme cholinesterase, the acetylcholinesterase (AChE) and the butyrilcholinesterase (BuChE). Increased activity of fractions of AChE has been associated with states of dementia in elderly patients and the loss of cognitive function in patients with Alzheimer's Disease. This work is proposed to study the expression of neuronal glucose transporter GLUT3 and activity of the enzyme acetylcholinesterase (AChE) in brain of young adult rats (84 days of life) who were breastfed in litters formed by 6 (control group) or 12 pups (undernourished group). The results showed that the weight of the brains of malnourished rats was significantly lower (P <0001, Student s t test) compared to controls. The blood glucose levels and the expression of GLUT3 in total cortical membranes was also reduced by malnutrition (P <0001, Student s t test). The activity of AChE in different brain homogenate showed a significant interaction (P = 0019, two-way ANOVA, Tukey s test) between nutritional status and the type of fraction of the homogenate, with a significant reduction (p< 0,05) in activity this enzyme in the brain homogenate in control group. These results show for the first time that malnutrition during the period of weaning reduces the expression of GLUT3 and vi that the increased activity of acetylcholinesterase associated with decreased in the expression of the main glucose transporter in the brain could contribute to cognitive deficits and changes in brain metabolic activity. / A desnutrição energético-protéica consiste em um dos problemas nutricionais mais graves que ocorrem em países em desenvolvimento. A deficiência energética e nutricional no início da vida pode ocasionar importantes alterações nas diferentes fases de desenvolvimento do sistema nervoso central, uma vez que os processos de hiperplasia, hipertrofia e mielinização ocorrem de forma mais intensa. O cérebro utiliza a glicose como seu principal substrato energético. No entanto, em certas condições fisiológicas, como durante a fase de amamentação ou durante o jejum prolongado, as células podem utilizar outros substratos a fim de suprir suas necessidades metabólicas. O transporte de glicose para as células nervosas requer a presença de proteínas transportadoras específicas denominadas GLUTs (glucose transporters) que efetuam o transporte de glicose através de difusão facilitada. Além dos transportadores de glicose, proteínas transportadoras de monocarboxilatos (MCTs) são responsáveis pelo influxo neuronal de lactato, sendo o MCT1 o principal responsável pelo fornecimento energético aos neurônios durante o processo de maturação do tecido nervoso. A acetilcolina é um dos principais neurotransmissores do sistema nervoso, sendo associada com a manutenção de processos de atenção, aprendizagem e memória. Na fenda sináptica a acetilcolina é degradada por enzimas colinesterásicas, a acetilcolinesterase (AChE) e a Butirilcolinesterase (BuChE). O aumento da atividade de frações da AChE vem sendo associada com estados de demência em pacientes idosos e com a perda da função cognitiva em pacientes com Doença de Alzheimer. O presente trabalho se propôs a estudar a expressão do transportador neuronal de glicose GLUT3 e a atividade da AChE no cérebro de ratos adultos jovens (84 dias de vida) que foram amamentados em ninhadas formadas por 6 (grupo controle) ou 12 filhotes (grupo desnutrido). Os resultados demonstraram que o peso dos cérebros dos ratos desnutridos foi significativamente menor (P<0,001, teste t de Student) comparado ao grupo controle. Os níveis glicêmicos e a expressão do GLUT3 nas membranas corticais totais foram também diminuídos pela desnutrição (P<0,001, teste t de Student). A atividade da AChE nos diferentes homogenados do cérebro mostrou uma interação significativa (P = 0,019, ANOVA two-way, Tukey teste) entre o estado iv nutricional e o tipo de fração do homogenado, apresentando uma redução significativa ( p< 0,05) na atividade desta enzima do homogenado de células em animais controle . Esses resultados mostram pela primeira vez que a desnutrição durante o período de amamentação diminui a expressão do GLUT3 e que o aumento da atividade da acetilcolinesterase associado com a diminuição da expressão do principal trnasportador de glicose para o cérebro poderia contribuir para déficits cognitivos e alterações da atividade metabólica cerebral.

Purpose: To determine whether cycle training of sedentary subjects would increase the expression of the principle muscle glucose transporters, six volunteers completed 6 wk of progressively increasing intensity stationary cycle cycling. Methods: In vastus lateralis muscle biopsies, changes in expression of GLUT1, GLUT4, GLUT5, and GLUT12 were compared using quantitative immunoblots with specific protein standards. Regulatory pathway components were evaluated by immunoblots of muscle homogenates and immunohistochemistry of microscopic sections. Results: GLUT1 was unchanged, GLUT4 increased 66%, GLUT12 increased 104%, and GLUT5 decreased 72%. A mitochondrial marker (cytochrome c) and regulators of mitochondrial biogenesis (peroxisome proliferator-activated receptor γ coactivator 1 α and phospho-5′-adenosine monophosphate-activated protein kinase) were unchanged, but the muscle hypertrophy pathway component, phospho-mammalian target of rapamycin (mTOR), increased 83% after the exercise program. In baseline biopsies, GLUT4 by immunohistochemical techniques was 37% greater in Type I (slow twitch, red) muscle fibers, but the exercise training increased GLUT4 expression in Type II (fast twitch, white) fibers by 50%, achieving parity with the Type I fibers. Baseline phospho-mTOR expression was 50% higher in Type II fibers and increased more in Type II fibers (62%) with training but also increased in Type I fibers (34%). Conclusion: Progressive intensity stationary cycle training of previously sedentary subjects increased muscle insulin-responsive glucose transporters (GLUT4 and GLUT12) and decreased the fructose transporter (GLUT5). The increase in GLUT4 occurred primarily in Type II muscle fibers, and this coincided with activation of the mTOR muscle hypertrophy pathway. There was little impact on Type I fiber GLUT4 expression and no evidence of change in mitochondrial biogenesis.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Physical and psychological stressors trigger activation of the hypothalamo-pituitary-adrenocortical (HPA) axis that leads to enhanced secretion of glucocorticoids e.g. cortisol. Moreover, chronic activation of this pathway may elevate oxidative stress that is linked to the onset of insulin resistance and cardiovascular diseases (CVD). Our laboratory previously found that oxidative stress increases flux through metabolic circuits such as the hexosamine biosynthetic pathway (HBP), in effect increasing its modification of target proteins post-transcriptionally with O-GlcNAc moeities. This in turn may alter protein function and contribute to the onset of myocardial insulin resistance and impaired contractile function. Since the underlying mechanisms linking chronic stress to cardiometabolic pathophysiology are poorly understood, we hypothesised that cortisol elicits myocardial oxidative stress, HBP activation, and decreased glucose uptake (due to attenuated glucose transport functionality) with detrimental outcomes, i.e. insulin resistance and apoptosis. To investigate this hypothesis we established an in vitro model using HL-1 cardiomyocytes, with which we evaluated the degree of O-GlcNAcylation and oxidative stress in response to a range of time-dose treatments with dexamethasone (synthetic glucocorticoid). Glucose transporter 4 (GLUT4) translocation to the sarcolemma was also assessed. In agreement with the literature, results suggest that GLUT4 translocation is significantly decreased subsequent to dexamethasone treatment. Although no significant differences were observed with regards to oxidative stress or O-GlcNAcylation, the data show that dexamethasone increased the latter with a maximal effect after two hours exposure to the 10-6 M dose. Although our results were not conclusive, the data suggest a potential novel link between dexamethasone exposure, HBP activation and decreased GLUT4 translocation. Based on our findings we propose that detrimental effects of chronic stress on the heart may be mediated by increased HBP flux. Given that glucocorticoid excess and GLUT4 dysregulation have been associated with insulin resistance (and related metabolic derangements and diseases), these results provide new targets for potential therapeutic agents. / AFRIKAANSE OPSOMMING: Fisiese sowel as psigologiese stressors veroorsaak die aktivering van die hipotalamiese-hipo seale-bynier (HHB) pad wat lei tot die verhoogde sekresie van glukokortikoïede soos kortisol. Kroniese aktivering van hierdie pad kan ook oksidatiewe stres verhoog wat weer tot insulienweerstandigheid en kardiovaskulêre siektes (KVS) kan lei. Navorsing uit ons laboratorium het voorheen bewys dat oksidatiewe stres 'n toename in vloei deur metaboliese paaie soos die heksoamine biosintetiese pad (HBP) kan veroorsaak deur die modi sering van teikenproteïene met O-GlcNAc motiewe. Dit kan weer proteïen funksie verander en bydra tot die ontstaan van miokardiale insulienweerstandigheid en verswakte kontraktiele funksie. Die onderliggende meganismes wat kroniese stres aan kardiometaboliese pato siologie verbind word nog nie goed verstaan nie, daarom is ons hipotese dat kortisol miokardiale oksidatiewe stres veroorsaak, die HBP pad aktiveer, en glukose opname verminder (deur die funksionele onderdrukking van glukose transport), wat nadelige uitkomste soos insulienweerstandigheid en apoptose tot gevolg kan hê. Om hierdie hipotese te ondersoek, is 'n in vitro model van HL-1 kardiomiosiete gebruik waarmee die graad van O-GlcNAsilering en oksidatiewe stres in reaksie op 'n reeks tyd-konsentrasie behandelings met deksametasoon (sintetiese glukokortikoïed), bepaal is. Glukose transporter 4 (GLUT4) translokasie na die sarkolemma is ook geasseseer. In ooreenstemming met die literatuur, is GLUT4 translokasie insiggewend onderdruk tydens deksometasoon behandeling. Alhoewel geen insiggewende verskille rakende oksidatiewe stres en O-GlcNAsilering gevind is nie, het ons data aangedui dat laasgenoemde deur deksametasoon vermeerder het na twee ure van blootstelling aan die 10-6 M konsentrasie. Alhoewel ons resultate geen afdoende bewys lewer nie, stel dit wel voor dat daar 'n potensiële verbintenis tussen deksametasoon behandeling en 'n afname in GLUT4 translokasie is. Gebasseer op ons bevindings, stel ons voor dat die nadelige e ekte van kroniese stres op die hart bemiddel kan word deur 'n toename in vloei deur die HBP. Gegewe dat 'n oormaat glukokortikoïede en GLUT4 wanregulering geassosieer is met insulien weerstandigheid (en verbandhoudende metaboliese veranderinge en siektes), verskaf hierdie resultate nuwe teikens vir potensiële terapeutiese ingrepe.

No description available.

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. / Chronic renal failure (CRF) is characterized by multiple homeostasis imbalances such as insulin resistance. However, few studies addressed the underlying mechanisms of the insulin resistance in CRF. Moreover, it is not known if the insulin resistance in CRF could lead to type II diabetes in predisposed patients. In 5/6th nephrectomised Sprague-Dawley (CD) rat model of CRF, we observed a correlation between the severity of the renal injury, evaluated by the serum creatinine level, and the hyperglycaemia, evaluated by the serum fructosamine level (R2 = 0.6982, p < 0.0001). However, this hyperglycemia is not observed on fasting. During a glucose tolerance test, we noticed an increase of the glycaemia in CRF rats (AUC 1.25 fold, p < 0.0001) comparing to controls. Insulin secretion of CRF rats was not significantly higher (AUC ≈1.30 fold, N.S.) during glucose challenge. Interestingly, despite more increase in insulin levels in CRF rats following a glucose bolus, the peripheral tissues did not show any increase in blood glucose uptake suggesting a defect in expression and/or function of glucose transporters in these rats. Indeed, we observed decreased expression of glucose transporters in the liver (≈0.60 fold, p < 0.01) and muscles (GLUT1 0.73 fold, p < 0.05 and GLUT4 0.69 fold, p < 0.01). Accordingly, there was a significant reduction in the insulin-dependent glucose uptake in the muscles of CRF rats compared to controls (≈0.63 fold, p < 0.0001). Since muscles are responsible for the majority of insulin-sensitive glucose transport, downregulation of GLUT4 could be associated with the insulin resistance observed in CRF. The modulation of the expression of several glucose transporters may contribute to insulin resistance in CRF, but other mechanisms could also be implicated. Despite this important perturbation of glucose transport, we did not observed any case of type II diabetes in our CD rat model. In a rat model of metabolic syndrome, the Zucker Leprfa/fa, CRF causes a strong hyperglycemia on fasting (1.5 fold, p < 0.0001). Furthermore, CRF Zucker showed an exacerbated glycemic response (AUC 1.80 fold, p < 0.0001) during glucose challenge. A strong insulin resistance in muscle was measured as the usual insulin dose (2mU/mL) was not enough to stimulate glucose uptake in Zucker rats with CRF. The same modulation of glucose transporters in the peripheral tissues was observed in both rat models. As opposed to CD rats, ≈30% (p < 0.05) of CRF Zucker rats’ showed presence of glucose in their urine. CRF by itself won’t lead to type II diabetes. However, when insulin resistance is already present when developing CRF, it could precipitate the onset of type II diabetes among these patients.