Type 2 diabetes mellitus (T2DM) is an increasing global health concern, currently affecting an estimated 382 million individuals. There is no cure for T2DM and the search for new and improved treatments is ongoing. Presently, various pharmacological regimens are available to treat T2DM, but with varied success. Thousands of traditional herbs are also used to treat T2DM, but mainly without scientific validation. The aim of this study was to assess the polyphenolic content, antioxidant capacity, as well as in vitro toxicity and hypoglycaemic activity of a commercial ‘antidiabetic’ tea mixture (Diabetea) and its individual constituents: Achillea millefolium L. (Yarrow), Agathosma betulina Bartl. & Wendl. (Buchu), Salvia officinalis L. (Sage), Taraxacum officinalis L. (Dandelion), Thymus vulgaris L. (Thyme), Trigonella foenum-graecum L. (Fenugreek) and Urtica urens L. (Nettle). All herbs were tested as crude extracts, prepared using hot water (HW) and dichloromethane (DCM). The total polyphenolic content of each extract was determined using the Folin-Ciocalteau and aluminium trichloride methods. The non-cellular antioxidant activity was assessed using 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods. The cell-based antioxidant activity was measured against p-chloranil-induced generation of reactive oxygen species (ROS) in Ea.hy926 cells, using the fluorescent dye, 2',7'-dichlorfluorescein-diacetate (DCFH-DA). The effect of each extract on the viability of C2C12 myotubes, Ea.hy926 endothelial cells and human lymphocytes (HL) was determined using sulforhodamine B (SRB). The in vitro hypoglycaemic activity was assessed against α-amylase and α-glucosidase activity using 3,5-dinitrosalicylic acid (DNSA) and p-nitrophenyl-α-D-glucopyranoside (p-NPG), respectively. The type of inhibition exerted on these enzymes was determined using the Michaelis-Menten enzyme kinetics model, expressed as mixed, competitive, non-competitive and uncompetitive. Glucose uptake activity was measured using the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) fluorescent analogue. T. vulgaris and S. officinalis had the highest amount of polyphenols of all extracts tested. The HW extracts of T. vulgaris and S. officinalis showed significant (p < 0.05) cell-free antioxidant activity and cell-based radical scavenging activity. In addition, U. urens (HW) also limited cell-based ROS generation (p < 0.05). The Diabetea extracts presented with poor antioxidant activities, of which some had a pro-oxidant effect on Ea.hy926 cells. The positive linear relationship between antioxidant activity and polyphenolic content was shown to be dependent on the solvent type used. All of the DCM extracts had low antioxidant activity and polyphenolic content. None of the extracts produced < 50% cell density at the concentrations tested (1.3 - 20 μg/mℓ). In general, the DCM extracts showed a greater decrease in cell density than the HW extracts. The Ea.hy926 cells were the least affected by the extracts in terms of decreased cell density. The DCM extract of U. urens inhibited α-amylase activity in a mixed manner, which was comparable to the percentage inhibition exerted by the commercial drug, acarbose. Both the HW and DCM extracts of U. urens caused a significant (p < 0.05) increase in glucose uptake into C2C12 myotubes. The HW extract of T. vulgaris had a significant (p < 0.05) inhibitory activity against α-glucosidase (mixed). It also caused the uptake of glucose into C2C12 myotubes, which was significantly (p <0.05) more active than insulin. S. officinalis (DCM extract) also inhibited α-glucosidase activity (p < 0.05) in a mixed manner. Its HW extract displayed potent hypoglycaemic potential by causing glucose uptake into C2C12 myotubes, which was more significant (p < 0.05) than the activity of the positive control, insulin. The DCM extract of A. betulina was active against α-glucosidase (non-competitive), which was comparable to the activity of acarbose. Its HW extract also showed a significant (p < 0.05) glucose uptake activity. Furthermore, the DCM extracts of T. officinalis, A. millefolium, Diabetea and HW extracts of T. foenum-graecum and T. officinalis also caused a significant (p < 0.05) increase in glucose uptake into C2C12 myotubes. This study provides evidence for the antidiabetic potential of T. vulgaris and S. officianlis, in terms of antioxidant capacity and potential to prevent of post-prandial hyperglycaemia and alleviate hyperglycaemia by mimicking the action of insulin. In addition, the organic preparation of U. urens is also a potent α-amylase inhibitor. All herbs tested in this study exerted some form of in vitro antidiabetic activity. The Diabetea mixture, as a traditional preparation, did not have a significant antidiabetic capacity. In vitro observations from this study do not support the use of Diabetea as an antidiabetic preparation and reveal that some of the individual extracts prove more efficacious than the herb mixture. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Pharmacology / MSc / Unrestricted

Positron emission tomography (PET) visualizes increased cellular [18F]fluorodeoxyglucose ([18F]FDG) uptake. Pulmonary hypertension (PH) is conceived of a proliferative disease of the lung vessels. Increased glucose uptake can be quantified as pulmonary [18F]FDG uptake via PET imaging. Because the angioproliferative mechanisms in PH are still in need of further description, the aim of the present study was to investigate whether [18F]FDG PET/CT imaging can elucidate these pathophysiologic mechanisms in different etiologies of PH. Patients (n = 109) with end-stage pulmonary disease being evaluated for lung transplant were included in this observational study. Mean standardized uptake value (SUVmean) of predefined regions of interest in lung parenchyma (LP), left (LV), and right ventricle (RV) of the heart, and SUVmax in pulmonary artery (PA) were determined and normalized to liver uptake. These SUV ratios (SUVRs) were compared with results from right heart catheterization (mean pulmonary artery pressure [mPAP], pulmonary vascular resistance [PVR]), and serum N-terminal pro-brain natriuretic peptide. Group comparisons were performed and Pearson correlation coefficients (r) were calculated. The [18F]FDG uptake ratios in LP, RV, RV/LV, and PA, but not in LV, were found to be significantly higher in both patients with mPAP ≥25 mm Hg (P = 0.013, P = 0.006, P = 0.049, P = 0.002, P = 0.68, respectively) and with PVR ≥480 dyn·s/cm5 (P < 0.001, P = 0.045, P < 0.001, P < 0.001, P = 0.26, respectively). The [18F]FDG uptake in these regions positively correlated also with mPAP, PVR, and N-terminal pro-brain natriuretic peptide. The SUVR of PA positively correlated with the SUVR of LP and RV (r=0.55, r=0.42, respectively). Pulmonary and cardiac [18F]FDG uptake in PET imaging positively correlated with the presence and severity of PH in patients with end-stage pulmonary disease. Increased glucose metabolism in the central PAs seems to play a certain role in terms of severity of PH. These results suggest that [18F]FDG-PET imaging can help understand the pathophysiology of PH as a proliferative pulmonary disease.

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Le diabète est reconnu comme un problème majeur de santé publique causant des conséquences humaines et économiques redoutables. La phytothérapie s’offre comme une nouvelle avenue thérapeutique pour le contrôle de la glycémie. Le grenadier, Punica granatum, a servi de remède contre le diabète dans le système Unani de la médecine pratiquée en Inde et au Moyen Orient. Des études ont démontré un effet hypoglycémiant des extraits de grenadier via divers mécanismes notamment par une amélioration de la sensibilité à l’insuline et la régénération des cellules béta-pancréatiques. Cependant, aucune étude n’a démontré à ce jour, l’effet de grenadier sur le transport de glucose dans le muscle, étape cruciale dans la régulation de l’homéostasie glucidique postprandiale. De plus, l’effet de la maturation sur le potentiel antidiabétique du fruit de grenadier n’a pas été étudié. Ainsi, le but de ce projet est d’évaluer l’effet antidiabétique des extraits de grenadier sur le transport de glucose dans les cellules musculaires C2C12 en fonction de la variété et du stade de maturation du fruit et d’élucider les mécanismes d’action. Le choix des variétés du grenadier tunisien (Espagnoule [EP] et Gabsi [GB]) a été orienté pour leur pouvoir antioxydant et leur consommation locale. Deux parties de la plante ont été utilisées, les fleurs et les fruits à 3 stades de maturation soit 2, 4 et 6 mois. Les résultats ont montré que seule la variété du grenadier Gabsi stimule significativement le transport de glucose par rapport au contrôle (DMSO), et ceci sans être toxique. Cet effet est plus prononcé au stade de fruit mûr (à 6 mois) que celui de la fleur. De plus, l’extrait de fleurs stimule la voie insulino-indépendante de l’AMPK et augmente le niveau d’expression des transporteurs spécifiques de glucose (GLUT-4). Par contre, l’extrait de fruits mûrs, en plus de ces deux mécanismes, active fortement aussi la voie insulino-dépendante de l’AKT. En conclusion, cette étude présente un nouveau mécanisme d’action antidiabétique de grenadier (plus particulièrement du fruit mûr) qui est dépendant de la variété. / Diabetes is a major public health problem worldwide with astounding human and economic consequences. The seed and the flower of pomegranate (Punica granatum), a native plant of Central Asia and the Mediterranean regions, exhibited a hypoglycaemic effect in in vivo studies. However, the underlying mechanisms have not yet been elucidated. The aim of this project was to evaluate the effect of the flower and the fruit (at 3 maturation stages) of pomegranate on glucose transport in skeletal muscle cells and to determine the molecular mechanisms involved in this effect. To accomplish this, we chose two varieties of pomegranate cultivated in Tunisia (Gabsi [GB] and Espagnoule [EP]), which have been shown to be highly consumed in that area and to possess high antioxidant activity. Differentiated C2C12 cells were treated for 18 hours with 80% ethanolic extract of the flowers and fruits (at 2, 4, and 6 months) of each variety. Our results showed that the Gabsi variety of pomegranate significantly enhances glucose uptake, without any toxicity. This effect is more pronounced in the ripe fruit (6 months) than in the flower. In parallel, the ripe fruit stimulated both the insulin-dependent pathway (Akt) and the insulin-independent pathway (AMPK), while the flower stimulated the latter only. In addition, both flower and ripe fruit treatment resulted in enhanced expression level of GLUT-4 glucose transporter in the muscle. Hence, these results suggest that regulation of glucose transport in skeletal muscle is one of the components involved in the anti-diabetic effect of Tunisian pomegranate.

Os hormônios tireoidianos atuam sobre o metabolismo dos diversos tecidos do organismo e participam da regulação do consumo de glicose pelas células. Estudos já evidenciaram que o T3 atua, dependendo do tipo celular, aumentando a expressão de algumas isoformas dos transportadores de glicose (GLUTs) e a translocação do GLUT4 para a membrana plasmática, melhorando, também, a captação de glicose (CGlic) em poucos minutos. Os mecanismos envolvidos nessas ações do T3, contudo, ainda não estão bem esclarecidos. O objetivo do presente estudo foi investigar as possíveis vias de sinalização envolvidas na ação aguda do T3 sobre a CGlic em células adiposas 3T3-L1. Nossos dados demonstraram que o T3 promove aumento na CGlic, com pico aos 10 min, retornando ao nível do controle após 30 min de incubação das células com o hormônio. Sugerimos que essa ação depende, ao menos, de duas vias de sinalização. Uma delas envolve a ativação das proteínas Src, PI3K e Akt. A outra, aparentemente, é iniciada a partir da membrana plasmática via integrina aVb3 / Thyroid hormones act on the metabolism of many tissues and participate in the regulation of glucose consumption by cells. Studies from this and other laboratories have demonstrated in muscle and adipose cells that T3 increases, in a short period (minutes), the expression of some glucose transporter (GLUTs) isoforms and GLUT4 translocation to the plasma membrane leading to an improvement of glucose uptake. However, the mechanisms involved in these T3 actions are still not clear. The aim of this study was to investigate the possible signaling pathways involved in the acute T3 action on glucose uptake in 3T3-L1 adipocytes. Our results have shown that T3 increases glucose uptake with a peak at 10 min returning to the control level after 30 min of the cell incubation with the hormone. We suggest that this action depends on at least two parallel signaling pathways. One involves the activation of Src, PI3K and Akt proteins, while the other involves another mechanism triggered by T3, apparently, from the plasma membrane at aVb3 integrin.

Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures.   The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z. / QC 20110608

Le diabète est reconnu comme un problème majeur de santé publique causant des conséquences humaines et économiques redoutables. La phytothérapie s’offre comme une nouvelle avenue thérapeutique pour le contrôle de la glycémie. Le grenadier, Punica granatum, a servi de remède contre le diabète dans le système Unani de la médecine pratiquée en Inde et au Moyen Orient. Des études ont démontré un effet hypoglycémiant des extraits de grenadier via divers mécanismes notamment par une amélioration de la sensibilité à l’insuline et la régénération des cellules béta-pancréatiques. Cependant, aucune étude n’a démontré à ce jour, l’effet de grenadier sur le transport de glucose dans le muscle, étape cruciale dans la régulation de l’homéostasie glucidique postprandiale. De plus, l’effet de la maturation sur le potentiel antidiabétique du fruit de grenadier n’a pas été étudié. Ainsi, le but de ce projet est d’évaluer l’effet antidiabétique des extraits de grenadier sur le transport de glucose dans les cellules musculaires C2C12 en fonction de la variété et du stade de maturation du fruit et d’élucider les mécanismes d’action. Le choix des variétés du grenadier tunisien (Espagnoule [EP] et Gabsi [GB]) a été orienté pour leur pouvoir antioxydant et leur consommation locale. Deux parties de la plante ont été utilisées, les fleurs et les fruits à 3 stades de maturation soit 2, 4 et 6 mois. Les résultats ont montré que seule la variété du grenadier Gabsi stimule significativement le transport de glucose par rapport au contrôle (DMSO), et ceci sans être toxique. Cet effet est plus prononcé au stade de fruit mûr (à 6 mois) que celui de la fleur. De plus, l’extrait de fleurs stimule la voie insulino-indépendante de l’AMPK et augmente le niveau d’expression des transporteurs spécifiques de glucose (GLUT-4). Par contre, l’extrait de fruits mûrs, en plus de ces deux mécanismes, active fortement aussi la voie insulino-dépendante de l’AKT. En conclusion, cette étude présente un nouveau mécanisme d’action antidiabétique de grenadier (plus particulièrement du fruit mûr) qui est dépendant de la variété. / Diabetes is a major public health problem worldwide with astounding human and economic consequences. The seed and the flower of pomegranate (Punica granatum), a native plant of Central Asia and the Mediterranean regions, exhibited a hypoglycaemic effect in in vivo studies. However, the underlying mechanisms have not yet been elucidated. The aim of this project was to evaluate the effect of the flower and the fruit (at 3 maturation stages) of pomegranate on glucose transport in skeletal muscle cells and to determine the molecular mechanisms involved in this effect. To accomplish this, we chose two varieties of pomegranate cultivated in Tunisia (Gabsi [GB] and Espagnoule [EP]), which have been shown to be highly consumed in that area and to possess high antioxidant activity. Differentiated C2C12 cells were treated for 18 hours with 80% ethanolic extract of the flowers and fruits (at 2, 4, and 6 months) of each variety. Our results showed that the Gabsi variety of pomegranate significantly enhances glucose uptake, without any toxicity. This effect is more pronounced in the ripe fruit (6 months) than in the flower. In parallel, the ripe fruit stimulated both the insulin-dependent pathway (Akt) and the insulin-independent pathway (AMPK), while the flower stimulated the latter only. In addition, both flower and ripe fruit treatment resulted in enhanced expression level of GLUT-4 glucose transporter in the muscle. Hence, these results suggest that regulation of glucose transport in skeletal muscle is one of the components involved in the anti-diabetic effect of Tunisian pomegranate.

Introduction: La ménopause est associée à l’insulino-résistance et augmente le risque de diabète de type 2 (DT2) chez les sujets sains. Cependant, peu d’informations existent à savoir comment la ménopause et l’activité physique peuvent influencer l’homéostasie du glucose chez des sujets insulino-résistants. Objectifs: Déterminer 1) l’effet du retrait des œstrogènes ovariens par ovariectomie sur l’homéostasie du glucose des rates ZDF (Zucker Diabetic Fatty; prédisposées au diabète de type 2) et 2) évaluer l’influence de l’activité physique volontaire sur ces réponses. Méthodologie: Vingt-quatre rates furent d’abord nourries et hébergées dans des cages conventionnelles les 28 premiers jours pour ensuite subir une ovariectomie (OVX, n=16) ou une opération simulée (SHAM-Inactive, n=8). Les rates ovariectomisées furent ensuite assignées au groupe entraîné volontairement dans une cage à roue (OVX-Active, n=8) ou demeurèrent sédentaires (OVX-Inactive, n=8) pendant les 44 jours suivants. Résultats: Au jour 56, la glycémie à l’état nourri fut significativement augmentée par l’ovariectomie (p<0,01) et ramenée au niveau initial chez les rates OVX-Active (p<0,01). L’ovariectomie diminua la captation de glucose induite par l’insuline dans le muscle de façon significative (0,63 ± 0,08 vs 1,13 ± 0,27 μmol•g-1•h-1). L’entraînement améliora la tolérance au glucose (p<0,01) ainsi que la prise de glucose induite par l’insuline dans le muscle (p<0,05). Conclusion: Le retrait des estrogènes ovariens par ovariectomie perturbe l’homéostasie du glucose chez les rates ZDF femelles, sans pour autant provoquer le diabète de type 2. L’activité physique a un effet bénéfique sur l’homéostasie du glucose malgré la perte d’estrogènes ovariens. / Introduction: Menopause is associated with insulin resistance and increased risks of type 2 diabetes in healthy human subjects. However, little is known about its effects on glucose homeostasis in insulin-resistant subjects. Aims: Our aim was to study 1) the effects of ovariectomy and 2) voluntary physical activity on glucose homeostasis in ZDF (Zucker diabetic fatty) female rats, a well-known animal model of insulin resistance and diabetes. Methodology: Twenty-four rats were fed and housed in standard cages during 28 days after which they either underwent an ovariectomy (Ovx) or a sham operation (SHAM-Inactive, n=8). The ovariectomized rats either engaged in voluntary wheel cage running (OVX-Active, n=8) or remained inactive (OVX-Inactive, n=8) for the following 44 days. Results: Fed glycaemia at day 56 was significantly increased by Ovx (p<0.01) and lowered back to control level in OVX-Active rats (p<0.01). Ovx significantly decreased insulin-stimulated muscle glucose uptake (0.63 ± 0.08 vs 1.13 ± 0.27 μmol•g-1•h-1). OVX-Inactive rats also showed increased triglyceride (p<0.001) and lower glycogen (p<0.001) contents in their liver whereas pancreatic insulin content was increased (p<0.05) as compared to SHAM-Inactive rats. Training markedly improved glucose tolerance (p<0.01) and insulin-stimulated muscle glucose uptake (p<0.05) as compared to SHAM-Inactive rats. Ovx-induced alterations in pancreatic insulin content (p<0.01) and liver glycogen (p<0.05) were improved by physical activity. Conclusion: Our data suggest that ovariectomy-induced loss of ovarian estrogens impairs glucose homeostasis in female ZDF rats without triggering overt type 2 diabetes. Physical activity improves glucose homeostasis despite the estrogen loss.

An der Spitze der Morbiditäts - und Mortalitätsstatistik steht weltweit das Metabolische Syndrom, bestehend aus androider Adipositas, pathologischer Glukosetoleranz, Dyslipidämie und arterieller Hypertonie, verbunden mit einer erhöhten Inzidenz atherosklerotischer Gefäßerkrankungen. Der Replikationsinitiator 1 (Repin1) wurde kürzlich als mögliches Kandidatengen für Adipositas sowie damit verbundene metabolische Funktionsstörungen in kongenen sowie subkongenen Rattenstämmen identifiziert. Ziel der Arbeit war es, den Einfluss von Repin1 auf den Fettzellstoffwechsel zu untersuchen. Hierfür wurde die Expression von Repin1 in 3T3– L1 Präadipozyten und differenzierten 3T3-L1 Adipozyten mittels siRNA Technologie stark vermindert, um so auf mögliche Funktionen des Proteins schließen zu können. Nachfolgend wurden Veränderungen des Zellstoffwechsels mittels Glukosetransport, Palmitataufnahme sowie Triglyceridgehalt der Adipozyten untersucht. Repin1 wird in der 3T3-L1 Zelllinie exprimiert und zeigt eine steigende Expression während der Adipogenese. Der Knockdown von Repin1 resultierte in kleineren Fettzellen mit geringerer basaler, jedoch verstärkter insulinstimulierter Glukoseaufnahme. Auch der Fettstoffwechsel zeigte sich alteriert: Neben einer reduzierten Palmitataufnahme war die Expression verschiedener Schlüsselgene der Fetttropfenfusion, des Glukose-sowie des Fetttransportes verändert. Fazit: Repin1 reguliert die Expression von Genen, die eine Rolle bei der Festlegung der Fettzellgröße und des basalen und Insulin-stimulierten Glukosetransports in Adipozyten spielen.:Inhaltsverzeichnis I Vorbemerkung II Wissenschaftlicher Anteil des Promovenden an der Publikation III Bibliographische Beschreibung IV Abkürzungsverzeichnis V I. Einleitung - Das metabolische Syndrom 1 A. Definition und Prävalenz 1 B. Klinische Relevanz 2 C. Pathophysiologie 3 D. Exogene und genetische Faktoren 7 II. Der Replikationsinitiator 1 9 III. Zielstellung der Arbeit 11 IV. Publikation 12 V. Zusammenfassung 19 VI. Supplemental Materials 24 VII. Literaturverzeichnis 32 VIII. Eigenständigkeitserklärung 38 IX. Curriculum vitae 39 X. Veröffentlichungen im Rahmen dieser Arbeit 41 XI.Danksagung 42