Objective: IGF1 is associated with metabolic parameters and involved in glucose metabolism. Low-IGF1 has been implicated in the etiology of glucose intolerance and subjects with pathological causes of either low- or high-IGF1 are at risk of diabetes. We hypothesized that both low- and high-IGF1 levels increase the risk of diabetes and aimed to assess the role of IGF1 in the risk of developing diabetes in a large prospective study. Design: An analysis of two prospective cohort studies, the DETECT study and SHIP. Methods: We measured IGF1 levels in 7777 nondiabetic subjects and assessed incident diabetes mellitus during follow-up. Results: There were 464 cases of incident diabetes during 32 229 person-years (time of follow-up in the DETECT study and SHIP: 4.5 and 5 years respectively). There was no heterogeneity between both studies (P>0.4). The hazard ratios (HRs) of incident diabetes in subjects with IGF1 levels below the 10th or above the 90th age- and sex-specific percentile, compared to subjects with intermediate IGF1 levels, were 1.44 (95% confidence interval (CI) 1.07–1.94) and 1.55 (95% CI 1.06–2.06) respectively, after multiple adjustment. After further adjustment for metabolic parameters, the HR for low-IGF1 became insignificant. Analysis of IGF1 quintiles revealed a U-shaped association of IGF1 with risk of diabetes. Results remained similar after exclusion of patients with onset of new diabetes within 1 year or with borderline glucose or HbA1c levels at baseline. Conclusions: Subjects with low- or high-IGF1 level are at increased risk of developing diabetes.

Einleitung: Die Untersuchung des regionalen zerebralen Glucosestoffwechsels mittels [18F]-2-Fluor-2-desoxy-D-glucose Positronen-Emissions-Tomographie ([18F]FDG-PET) ist ein etabliertes Verfahren der molekularen Bildgebung in der Diagnostik kognitiver und affektiver Störungen. Zwischen verschiedenen Untersuchungen kann es zu intra- und interindividuellen Unterschieden im Vigilanzniveau kommen. Das Ziel dieser ersten Machbarkeitsstudie war die Untersuchung des Zusammenhangs von aktuellem Vigilanzniveau und regionalem Glucosestoffwechsel während der [18F]FDG-PET. Methoden: 14 ältere Patientinnen und Patienten mit depressiver Episode oder leichter kognitiver Beeinträchtigung (MCI, mild cognitive impairment) wurden mit simultaner Elektroenzephalographie und [18F]FDG-PET unter Ruhebedingungen untersucht. Der Zusammenhang von Vigilanzniveau und regionalem Glucosestoffwechsel wurde mittels voxelweiser einfacher linearer Regression analysiert. Ergebnisse: Der Hauptbefund war eine Zunahme des regionalen zerebralen Glucosestoffwechsels mit abnehmendem Vigilanzniveau während der [18F]FDG-PET-Untersuchung in räumlich ausgedehnten frontalen und temporalen Kortizes. Diskussion: Vigilanzbezogene Veränderungen des Glucosestoffwechsels finden sich in vergleichbaren Hirnregionen und Effektstärken wie Veränderungen des Glucosestoffwechsels bei Patientinnen und Patienten mit depressiver Störung oder MCI gegenüber Gesunden. Der Einfluss des Vigilanzniveaus auf den Glucosestoffwechsel während der [18F]FDG-PET-Untersuchung sollte in kontrollierten Studien gesunder Personen validiert werden.

Indiana University-Purdue University Indianapolis (IUPUI) / Literature has demonstrated the ability of human adipose tissue to generate large amounts of lactate. However, it is not understood why adipose tissue produces lactate, how the production of lactate is regulated, and what potential benefit this has to the adipocyte or the organism. We first characterized a human model of adipogenic differentiation with minimal donor to donor variability to assess metabolic changes associated with mature adipocytes compared to their precursors. Indeed, similar to what was observed in human clinical studies, the differentiated adipocytes demonstrated increased lactate production. However, the differentiated adipocytes compared to their precursors (preadipocytes or ASCs) demonstrate an aerobic glycolysis-like (also called Warburg effect-like) increase in glycolysis characterized by a 5.2 fold increase in lactate production in normoxic conditions (atmospheric oxygen tension). Remarkably, this increase in lactate occurred even though the differentiated adipocytes simultaneously demonstrate an increase in oxidative capacity. This low fraction of oxidative capacity coupled with increased lactate production indicated regulation of oxidative rates most likely at the point of pyruvate conversion to either acetyl-CoA (oxidative metabolism) or lactate (glycolytic metabolism). To investigate the potential regulation of this metabolic phenotype, PDK isoform expression was assessed and we found PDK 1 and 4 transcript and protein elevated in the differentiated cells. Non-selective pharmacologic inhibition of the PDKs resulted in decreased lactate production, supporting a regulatory role for PDK in modulation of the observed Warburg effect. PDK inhibition also resulted in increased ROS production in the adipocytes after several hours of treatment and a decrease in cell viability when PDK inhibition was carried out over 36 hours. The resulting loss in viability could be rescued by antioxidant (Tempol) treatment, indicating the decrease in viability was ROS mediated. Similar to what is seen in cancer cells, our data demonstrate that differentiation of human adipocytes is accompanied by a PDK-dependent increase in glycolytic metabolism (Warburg effect) that not only leads to lactate production, but also seems to protect the cells from increased and detrimental generation of ROS.

The prevalence of obesity is dramatically increasing and thus constitutes a major risk factor for developing chronic diseases such as type 2 diabetes, dyslipidemia, cardiovascular diseases, and certain forms of cancer. High-caloric nutrition and a lack of physical activity are the main contributing factors for this global epidemic. Estrogen receptors (ERs) are recognized to be involved in many processes related to the control of energy homeostasis. In my studies, I investigated the impact of estrogens (17beta-estradiol (E2)) on energy homeostasis. Special emphasis was given to the effects of two synthetic ER subtype-selective agonists, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), to determine to what extend the two distinct ER subtypes are involved in the underlying molecular mechanisms. Because of its estrogenic activity and also its widespread use as a nutritional supplement the influence of the isoflavone genistein (Gen) was examined. For this purpose two different female rat models were used: Wistar rats with nutrition-induced obesity and leptin resistant Zucker diabetic fatty (ZDF) rats. In both experiments, the animals were ovariectomized (OVX) and treated with vehicle (untreated controls) or the estrogenic compounds. The most important finding was that treatment of OVX animals with Beta enlarges soleus muscle fiber sizes in both animal models compared to untreated OVX animals. This anabolic effect may in turn improve the muscle/fat ratio of the body that enhances muscular uptake and utilization of fuels. By contrast, in the gastrocnemius muscle of OVX ZDF rats substitution with Alpha increased expression and distribution of the insulin-dependent glucose transporter 4 (GLUT4). Consequently, systemic insulin sensitivity in both animal models was improved by treatment with estrogenic compounds compared to untreated OVX animals. The strongest effect was observed in E2-treated rats that indicate an additive effect through activation of both pathways. In all OVX rats, treatment with either ER subtype-selective agonist showed an anti-lipogenic effect in adipose tissue, liver, and skeletal muscle of nutrition-induced obese Wistar rats in comparison to OVX animals without treatment. Decreased visceral fat mass, adipocyte sizes, serum leptin levels, triglyceride accumulation in liver and muscle as well as mRNA expression of genes that are involved in lipo-/adipogenesis reflected this. Therefore, the lower visceral fat mass as well as decreased accumulation of triglycerides in non-adipose tissues such as liver and skeletal muscle most likely contributes to the improved insulin sensitivity in such treated animals. Gen exerted effects similar to those of the ER beta-selective agonist (except on adipose tissue in Wistar rats). Especially, the similar ability to induce anabolic activity in the soleus muscle might be highly relevant. Gen-treated animals might have a more effective utilization of fuels compared to untreated OVX animals because they showed a lower TG content in muscle and liver as well as improved glucose metabolism. In conclusion, because of my studies and the fact that ER beta signaling is not involved in proliferation of uterus and mammary gland, an effective way to treat obesity and co-morbidities in postmenopausal women might be substances that only activate ER beta. A combination with physical activity may support the therapy of obesity and co-morbidities. The isoflavone Gen is able to activate both ER-subtypes. This compound is already placed on the market for treatment of postmenopausal complaints, although adverse effects of Gen cannot be excluded so far (e.g., increased risk of breast cancer). However, Gen might be a natural alternative – not only to the conventional hormone replacement therapy, but also as a strategy for treatment of obesity and co-morbidities – that deserves further research with respect to these new data. / Die dramatisch zunehmende Prävalenz der Adipositas und das damit verbundene Risiko für Folgeerkrankungen wie Diabetes mellitus, Hypertonie, Dyslipidämie und koronare Herzkrankheiten stellt eine große Herausforderung für das Gesundheitswesen dar. Als Hauptursache wird ein chronisches Missverhältnis der Energiehomöostase aufgrund permanenter Überernährung und Bewegungsmangel postuliert. Estrogene beeinflussen den Glukose- und Lipidstoffwechsel und sind somit in die Regulation des Energiehaushaltes involviert. Estrogene vermitteln ihre Effekte über zwei Estrogenrezeptor (ER)-Subtypen, den ER alpha und den ER beta. Ziel der vorliegenden Arbeit war es mittels tierexperimentellen Studien den Einfluss von Estrogenen, speziell 17beta-Estradiol, auf den Energiehaushalt zu untersuchen. Um einen tieferen Einblick in die zugrundeliegenden molekularen Mechanismen zu erhalten, wurden zwei Subtyp-selektive ER-Agonisten, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), synthetischer Herkunft eingesetzt. Aufgrund der estrogenen Aktivität und der Verfügbarkeit als Nahrungsergänzungsmittel wurde des Weiteren der Einfluss des Isoflavons Genistein untersucht. Für die Studien wurden zwei Tiermodelle genutzt: zum einen weibliche Wistar-Ratten mit ernährungsinduzierter Adipositas und zum anderen weibliche leptinresistente „Zucker diabetic fatty“ (ZDF)-Ratten. Die Tiere wurden ovarektomiert (OVX) und entweder mit einem Vehikel (unbehandelte Kontrolltiere) oder mit der entsprechenden estrogenen Substanz behandelt. Die interessanteste Erkenntnis war, dass im Vergleich zu unbehandelten OVX-Tieren beider Tiermodelle die Behandlung mit Beta zur Vergrößerung der Faserquerschnitte im Soleusmuskel führte. Dieser anabole Effekt könnte die muskuläre Aufnahme und Verwertung von Brennstoffmolekülen verbessern und sich insgesamt positiv auf die Körperzusammensetzung auswirken. Den stärksten Effekt hinsichtlich einer erhöhten Expression und Translokation des insulinabhängigen Glukosetransporters 4 (GLUT4) in die Zellmembran des Gastrocnemiusmuskels zeigte sich dagegen durch die Behandlung von OVX ZDF-Ratten mit Alpha. Im Endergebnis zeigten die Tiere beider Modelle durch die Behandlung mit estrogenen Substanzen eine verbesserte systemische Insulinsensitivität im Vergleich zu unbehandelten Kontrolltieren. E2-behandelte Tiere tolerierten die Glukose am besten und lassen einen additiven Effekt aufgrund der Aktivierung beider Signalwege vermuten. Im Vergleich zu unbehandelten OVX Wistar-Ratten führte die Behandlung mit E2 oder mit jeweils einem der beiden ER-Subtyp-selektiven Agonisten zu einer geringeren viszeralen Fettmasse, kleineren Fettzellen, niedrigeren Leptinspiegeln im Serum und geringeren Triglyzeridwerten in Leber und Muskel. Auf der Ebene der Genexpression waren zudem geringere mRNA-Spiegel von lipo- und adipogenen Genen messbar. Somit scheinen beide ER-Subtypen in die antilipogene Wirkung von E2 involviert zu sein. Sowohl die reduzierte viszerale Fettmasse als auch die geringere Anreicherung von Triglyzeriden in Leber und Muskel tragen sehr wahrscheinlich ebenfalls zur verbesserten Insulinsensitivität bei. Die Behandlung von OVX Tieren mit Gen führte zu ähnlichen Ergebnissen wie die Behandlung mit Beta. Eine alleinige Ausnahme stellte das Fettgewebe dar, da hier eine Gen-Behandlung keine antilipogenen/-adipogenen Effekte zeigte. Speziell die Fähigkeit von Gen ebenfalls anabol zu wirken, könnte die molekulare Grundlage sein, weshalb Gen-behandelte Tiere im Vergleich zu unbehandelten Tiere eine verbesserte Toleranz gegenüber Glukose und eine geringere Anreicherung von Triglyzeriden in Muskel und Leber zeigten. Der ER beta ist nicht in die estrogenvermittelte Proliferation von Uterus und Brustdrüse involviert. Vor diesem Hintergrund lassen meine Ergebnisse vermuten, dass eine Behandlung mit ER beta-selektiven Substanzen eine effektive Möglichkeit darstellt, um Adipositas und deren Folgeerkrankungen in postmenopausalen Frauen zu behandeln, ohne deren Risiko für estrogenabhängige Krebsformen zu erhöhen. Eine Kombination mit regelmäßiger körperlicher Aktivität könnte die Erfolge bei der Behandlung von Adipositas und deren Folgeerkrankungen noch maximieren bzw. eine geringere Dosierung der verwendeten Substanz bei gleichbleibendem Behandlungserfolg ermöglichen. Das Isoflavon Gen mit seiner Fähigkeit beide ERs zu aktivieren ist eine bereits auf dem Markt befindliche Substanz und wird zur Behandlung von postmenopausalen Beschwerden eingesetzt, obwohl mögliche negative Effekte (z.B. ein erhöhtes Brustkrebsrisiko) noch nicht abschließend geklärt sind. Falls diese Risiken von Gen ausgeräumt werden können, könnte diese Substanz eventuell eine kostengünstige Alternative darstellen, um sowohl postmenopausale Beschwerden als auch Adipositas und deren Folgekrankheiten zu behandeln.

Nutrient sensing kinases lie at the heart of cellular health and homeostasis, allowing cells to quickly adapt to changing environments. Target of Rapamycin (TOR) and PAS kinase (PASK, or PASKIN) are two such nutrient kinases, conserved from yeast to man. In yeast, these kinases each have paralogs. The two TOR paralogs in yeast mimic the mammalian TORC1 and TORC2 complexes, except both Tor1 and Tor2 may contribute to TORC1 or TORC2 function. The two PAS kinase paralogs are paired with the TOR paralogs, meaning that both Psk1 and Psk2 regulate TORC1, while Psk2 suppresses a temperature-sensitive allele of Tor2. Herein we review the evolutionary models for these paralogs, their function in yeast and mammalian cells, as well as the overlapping function of PAS kinase and TOR. We also use Rice University’s Direct Coupling Analysis algorithms to analyze co-evolutionary relationships and identify potential interaction sites between PAS kinase and several of its substrates.

Mutations in the insulin-like growth factor-I (IGF-I) receptor (IGF1R) have been associated with prenatal and postnatal growth retardation. However, little is known about potential effects of mutations in the IGF1R on carbohydrate homeostasis. Methods: We investigated clinical, endocrine and metabolic parameters in four family members carrying a novel IGF1R mutation (p.Tyr387X): an 18-year-old male (index case), his sister and two paternal aunts. Results: All family members showed a variable degree of impairment in prenatal growth, with birth weight standard deviation scores (SDS) between –1.65 and –2.37 and birth length SDS between –1.78 and –3.08. Their postnatal growth was also impaired, with height SDS between –1.75 and –4.86. The index case presented high IGF-I levels during childhood and adolescence and delayed bone age. The index case and his two paternal aunts had impaired glucose tolerance (IGT) associated with a variable degree of alterations in insulin sensitivity and secretion. In contrast, the index case’s sister, who had had IGT during pregnancy, showed normal glucose metabolism but reduced insulin sensitivity. Conclusion: This is the first study showing an association between a novel IGF1R mutation and a variable degree of alterations in prenatal and postnatal growth and in carbohydrate metabolism.

Les nucléotides et nucléosides sont des molécules essentielles à la vie. Outre leurs fonctions intracellulaires, ils jouent un rôle dans la communication intercellulaire. Les nucléotides et nucléosides sont libérés dans l’espace extracellulaire par différents mécanismes et ensuite rapidement métabolisés par des ecto-nucléotidases. Ils exercent leurs effets paracrines et/ou autocrines en activant des récepteurs présents à la surface membranaire des cellules. Les récepteurs P1, au nombre de quatre (A1, A2A, A2B et A3), sont activés par l’adénosine. Les récepteurs P2X1-7 ont une activité intrinsèque de canal ionique et sont essentiellement activés par l’ATP. Les récepteurs P2Y possèdent sept domaines transmembranaires et sont couplés à des protéines G. A ce jour huit sous-types ont été identifiés :P2Y1,2,4,6,11,12,13,14. Ces récepteurs sont activés par des nucléotides adényliques (ATP et ADP) et/ou uridyliques (UTP, UDP et UDP-glucose). Les récepteurs P1 et P2 modulent l’activité de multiples processus biologiques :système immunitaire (A2A, P2X7, P2Y11,…), agrégation plaquettaire (P2Y1, P2Y12, P2X1), tonus vasculaire, angiogenèse,…<p><p>Notre laboratoire a identifié et caractérisé plusieurs récepteurs P2Y :P2Y4, P2Y6, P2Y11 et P2Y13. Ce dernier est activé par l’ADP et est couplé à une protéine Gi. L’abondance du transcrit P2Y13 murin est caractérisée par l’ordre suivant :rate >> pancréas > foie = cerveau. Afin de déterminer son rôle physiologique, nous avons généré une lignée de souris invalidées pour le récepteur P2Y13. Après avoir validé l’inactivation du gène P2Y13 dans ces souris, nous avons analysé leur phénotype. Les souris P2Y13-/- ne présentent pas d’anomalie évidente :elles sont viables, fertiles et se développent normalement. Etant donné le profil d’expression de ce récepteur, nous avons analysé leur système immunitaire, en particulier les cellules dendritiques (DC). <p>In vivo, l’invalidation du récepteur P2Y13 ne semble pas avoir d’impact sur les réponses inflammatoires (choc septique, infiltration de neutrophiles, test à la formaline) et auto-immunes (uvéorétinite expérimentale). In vitro, nous avons montré que l’ADPβS induit une mobilisation de calcium cytoplasmique dans les DC spléniques et qu’il stimule l’endocytose d’antigènes par celles-ci. L’utilisation de DC transgéniques a permis d’exclure l’implication du récepteur P2Y13 et a montré que ces effets sont médiés par le récepteur P2Y12 qui est également activé par l’ADPβS. Ces observations suggèrent qu’il serait intéressant d’analyser le système immunitaire des souris P2Y12-/-, en particulier les réponses immunes dépendantes des DC. D’autre part, ce travail a débouché sur la mise en évidence d’un effet anti-inflammatoire médié par le récepteur de l’adénosine A2B dans les DC dérivées de la moelle osseuse. Enfin, nous avons récemment mis en évidence un rôle potentiel du récepteur P2Y13 dans le métabolisme des glucides et des lipides. Nous avons observé que les souris P2Y13-/- produisent plus d’insuline en réponse à une injection de glucose que les souris contrôles tandis que leur glycémie ne semble pas altérée. De plus, les souris P2Y13-/- sous régime riche en graisses reproduisent 3 caractéristiques du syndrome métabolique chez l'homme :surpoids, dyslipidémie (augmentation des triglycérides et du non HDL-cholestérol) et hyperinsulinémie.<p><p>Notre travail de thèse débouche donc sur deux conclusions et une perspective :<p>•\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: A westernized diet rich in saturated fats and sugars, together with a sedentary lifestyle, has contributed to the dramatic increase in obesity during the last decade (Zimmett et al, 2001; Wild et al, 2004). Obesity is associated with dyslipidemia and insulin resistance which are major risk factors for the development of type 2 diabetes (T2D) (Zimmet et al, 2001, Kahn et al, 2006; Schröder et al, 2007). High-fat feeding in rodents induces symptoms similar to the human metabolic syndrome without progression to T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). The addition of fructose to a high-fat diet exacerbates the insulin resistance and leads to impaired pancreatic function of insulin secretion and glucose intolerance (Basciano et al, 2005; Stanhope et al, 2009). Aims: The aim of this study was to establish the effect of a high-fat and sucrose/fructose diet on glucose metabolism, the development of insulin resistance and β-cell dynamics. Methods: Weanling Wistar rats were randomized into two study groups; study one over an experimental period for three months and study two for twelve months. Each study consisted of a control group that received standard rat chow and water; and two experimental groups receiving either a high-fat diet and water (HFD) or a café diet consisting of HFD with the addition of 15% sucrose/fructose (CFD). Fasting glucose and insulin concentrations, intravenous glucose tolerance test (IVGTT), glucose stimulated insulin secretion rates and 2-deoxy-[3H]-D-glucose uptake in muscle, liver and fat were measured. The pancreata were harvested for immunohistochemical labeling of β-cells (insulin), α-cells (glucagon), GLUT2 (glucose transport) and MIB5 (proliferation). Samples of the pancreata were also collected for electron microscopy. Results and discussion: Feeding Wistar rats a CFD induced obesity, insulin resistance and glucose intolerance. By twelve months the rats had an impaired glucose response with increased IVGTT peak values, area under the curve (AUC) values and glucose clearance rates. Concomitantly, the glucose stimulated insulin secretion rate (GS-ISR) was attenuated. Stimulated glucose disposal as measured by 2-deoxy-[3H]-D-glucose uptake was reduced in muscle and adipose tissue at three months. By twelve months, due to the age of the rats, stimulated glucose uptake declined compared to three months with no difference between groups. After three months the diets had no observable effect on the islets using light microscopy. However, by twelve months morphological changes were observed in both the HFD and CFD groups. In the HFD group large hypertrophied irregular islets with fibrous changes were observed. In the CFD group these morphological changes were more prominent with fibrous segregation and disruption of the normal endocrine arrangement. In addition, the presence of inflammatory cells within the affected islets is consistent with T2D. Conclusion: High-fat diet fed to Wistar rats induced obesity, abdominal adiposity and insulin resistance. The addition of sucrose/fructose to a high-fat diet exacerbated the insulin resistance and resulted in glucose intolerance and mild hyperglycemia. Morphological changes in the large islets were observed which are consistent with the development of T2D. / AFRIKAANSE OPSOMMING: Inleiding: ‘n Verwesterde dieët, ryk aan versadigde vette en suikers tesame met 'n passiewe lewenstyl, het bygedra tot die dramatiese verhoging in vetsug gedurende die laaste dekade (Zimmett et al, 2001; Wild et al, 2004). Vetsug word met dislipidemie en insulienweerstandigheid geassosieer wat hoof risikofaktore is vir die ontwikkeling van tipe 2 diabetes (T2D) (Zimmet et al, 2001; Kahn et al, 2006; Schröder et al, 2007). Hoë-vet voeding in knaagdiere induseer simptome soortgelyk aan menslike metaboliese sindroom sonder die ontwikkeling van T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). Die byvoeging van fruktose tot 'n hoë-vet dieët vererger insulienweerstandigheid en lei tot verswakte pankreas funksie, insuliensekresie en glukoseintoleransie (Basciano et al, 2005; Stanhope et al, 2009). Doelwitte: Die doelwitte van die studie was om die effek van hoë-vet en sukrose/fruktose voeding op glukosemetabolisme, die ontwikkeling van insulienweerstandigheid en β-sel dinamika te bepaal. Metodes: Gespeende Wistar rotte was in twee groepe gerandomiseer; studie een oor ʼn tydperk van drie maande en studie twee oor ʼn tydperk van twaalf maande onderskeidelik. Elke studie het 'n kontrole groep met standaard rot kos en water (control); en twee experimentele diëte wat of ʼn hoë-vet dieët en water (HFD) of 'n kafeedieët groep wat die HFD met die byvoeging van 15% sukrose/fruktose in hul drink water (CFD) ontvang. Fastende glukose en insulien, binneaarse glukose toleransie toets (IVGTT), glukose gestimuleerde insulien sekresie tempo en 2-deoxi-[3H]-D-glukose opname in spier, lewer en vet is gebruik om die effek van die dieët op glukosemetabolisme te bepaal. Die pankreata is uitgehaal vir immunohistochemiese identifisering van β-selle (insulien), α-selle (glukagoon), GLUT2 (glukose transport) en MIB5 (proliferasie). Monsters van die pankreata was ook vir elektronmikroskopie versamel. Resultate en bespreking: Voeding van ʼn CFD aan Wistar rotte induseer vetsug, insulienweerstandigheid en glukose-intoleransie Teen twaalf maande toon die rotte 'n verswakte respons tot glukose met verhoogde IVGTT piekwaardes, AUC waardes en glukose opruimingswaardes. Terselfdetyd is die glukose gestimuleerde insuliensekresie tempo (GS-ISR) ook verswak. Gestimuleerde glukose opruiming, soos deur 2-deoxi-[3H]-D-glukose opname bepaal, was verlaag in spier en vetweefsel teen drie maande. Teen twaalf maande, weens die ouderdom van die rotte, is die gestimuleerde glukose opname verlaag in vergelyking met drie maande sonder 'n verskil tussen groepe. Na drie maande kon geen sigbare morfologiese verskille met ligmikroskopie tussen die diëte waargeneem word nie. Teen twaalf maande is morfologiese verskille waargeneem in beide die HFD en die CFD groepe. In die HFD groep is groot hipertrofiese onreëlmatige eilande met fibrotiese verandering waargeneem. In die CFD groep was die morfologiese verandering meer gevorder met fibrotiese onderverdeling en ontwrigting van die normale endokriene rangskikking. Die teenwoordigheid van inflammatoriese selle in die geaffekteerde eilande is verenigbaar met T2D. Afleiding: Die voer van 'n hoë-vet dieët aan Wistar rotte veroorsaak vetsug, abdominale adipositeit en insulienweerstandigheid. Die byvoeging van sukrose/ fruktose tot die hoë-vet dieët vererger die insulienweerstandigheid en veroorsaak glukoseintoleransie en matige hiperglukemie. Morfologiese veranderings in die groter eilande was verenigbaar met T2D.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: Athrixia phylicoides is an aromatic, indigenous shrub with high antioxidant content and numerous indigenous medicinal properties inferred by ingestion of an herbal brew of the plant. Commercialization of “bush tea” (derived from A. phylicoides) holds economic and developmental potential for indigenous communities provided the safety and efficacy of the herbal tea is established. Recently A. phylicoides has been shown by McGaw et al. (2007) to have similar antioxidant activity to Rooibos tea, and a unique, new flavonol (i.e. a polyphenolic antioxidant plant metabolite) 5-hydroxy-6,7,8,3′,4′,5′-hexamethoxyflavon-3-ol, unique to A. phylicoides, was isolated by Mashimbye et al. in 2006. With changes in the socio-economic climate and a new trend in merging Western lifestyle with traditional practices, new interest has been shown in herbal/natural remedies. Study Aim: The aim of this study was to firstly, determine the in vitro effect of A. phylicoides aqueous extract on glucose metabolism in cell lines that mimic the three key organs implicated in glucose homeostasis. Secondly, the study aimed to determine the potential ex vivo antioxidant and anti-inflammatory effect of the extract in pancreatic β-cells and peripheral mononuclear cells respectively. Methods: Leaves and fine twigs of A. phylicoides were processed into an aqueous extract. C2C12, Chang and 3T3-L1 cells were cultured under standard conditions and acutely exposed to increasing concentrations of extract and water vehicle, as well as 1 μM insulin and metformin as positive controls. Glucose uptake from 8 mM glucose culture media was determined using a fluorimetric oxidase method. Radioactive 14C-glucose oxidation to 14CO2 and determination of glycogen content of cells were used to assess the fate of intracellular glucose. RT-PCR was used to assess the extract effect on insulin-signalling gene expression. The antioxidative effect of A. phylicoides extract in pancreatic β-cells isolated from Wistar rats was determined by measuring nitric oxide (NO) production in response to hyperglycemic conditions. NO was labelled with diaminofluorocein diacetate and fluorescence was measured using flow cytometry. Insulin secretion of pancreatic β- cells was measured using radio-immuno assay. The anti-oxidative effect of the extract in lipopolysaccharide-stimulated peripheral mononuclear cells isolated from Wistar rats was determined by measuring the production of TNF-α using an ELISA kit. Results: C2C12 myocytes showed maximal increased glucose uptake at the 0.05 μg/μl extract concentration (228.3% ± 66.2, p<0.001). In Chang cells, A. phylicoides extract maximally increased the amount of glucose taken up at the 0.05 μg/μl concentration (134.5% ± 2.5, p<0.05). In 3T3-L1 cells, the extract maximally increased the amount of glucose taken up at the 0.025 μg/μl concentration (143.5% ± 10.3, p<0.001). An extract-induced increase in insulin receptor and glucose transporter four expression was seen in C2C12 myocytes. The oxidation of 14C-glucose to 14CO2 by C2C12 myocytes was maximally increased following acute exposure to the extract at 0.1 μg/μl (2919.3 fmol/1x10^6 cells ± 428, p<0.01). The oxidation of 14C-glucose to 14CO2 by Chang cells was maximally increased following acute exposure to extract at 0.1 μg/μl (4476.7 fmol/1x10^6 cells ± 1620, p<0.05); as seen in the C2C12 cells. A. phylicoides extract increased glycogen storage at all three concentrations tested in Chang cells, but maximally at the 0.025 μg/μl concentration (13.6 μg/1x10^6 cells ± 0.7, p<0.05). A. phylicoides extract did not have any measurable effect on the oxidative status of β-cells or the anti-inflammatory status of peripheral mononuclear cells. The extract did show an increase in first phase insulin secretion of β-cells in hyperglycemic conditions, although it was not significant. Conclusion: Athrixia phylicoides aqueous extract stimulates in vitro glucose uptake and metabolism in an insulin-mimetic manner, suggesting that this extract could potentially be beneficial to type two diabetics as an adjunct therapy. / AFRIKAANSE OPSOMMING: Inleiding: Athrixia phylicoides is 'n aromatiese, inheemse struik met 'n hoë antioksidant inhoud. Vele tradisionele medisinale eienskappe is gekoppel aan die ingestie van 'n kruie brousel van die plant, wat ook bekend as “bostee” is. Kommersialisering van “bostee” hou ekonomiese en ontwikkelings potensiaal in vir inheemse gemeenskappe mits die veiligheid en effektiwiteit van die kruietee bevestig kan word. McGaw et al. (2007) het onlangs bevind dat A. phylicoides se antioksidant aktiwiteit vergelykbaar is met die van rooibostee. 'n Unieke nuwe flavonol ('n polifenoliese antioksidant plant metaboliet) 5-hydroksie-6,7,8,3′,4′,5′-hexamethoksieflavon-3-ol, eie aan A. phylicoides, is deur Mashimbye et al. in 2006 geïsoleer. Met veranderings in die sosio-ekonomiese klimaat en 'n nuwe tendens om die westerse lewenstyl met tradisionele gebruike aan te vul word nuwe belangstelling in kruie/natuurlike rate ondervind. Studie Doelwitte: Die doelwitte van hierdie studie was eerstens om die in vitro effek van A. phylicoides waterekstrak op die glukosemetabolisme van drie sellyne wat die sleutel organe naboots wat glukosehomeostase beheer, te bepaal. Tweedens, is die potensiële ex vivo antioksidant en anti-inflammatoriese effek van die ekstrak op pankreatiese β-selle en perifere mononuklêere-selle onderskeidelik ondersoek. Metodes: n Waterige ekstrak is van die blare en fyn takkies van A. phylicoides berei. C2C12, Chang and 3T3-L1 selle is gekultuur onder standaard kondisies en akuut blootgestel aan stygende ekstrakkonsentrasies. Water het as kontrole gedien, met 1 μM insulien en metformien as positiewe kontroles. Glukose opname vanuit 8 mM glukose kultuurmedia is bepaal deur 'n fluorimetriese oksidase metode. Radioaktiewe 14C-glukose-oksidasie na 14CO2 en die bepaling van die glukogeen inhoud van selle is gebruik om die lot van intrasellulêre glukose te bepaal. RT-PKR is gebruik om die effek van die ekstrak op die insulien-seinpad geen-uitdrukking te ondersoek. Die antioksidant effek van A. phylicoides ekstrak in pankreatiese β-selle geïsoleer van Wistar rotte, is bepaal deur stikstofoksied (NO) produksie na aanleiding van hiperglukemiese kondisies. NO is met diaminofluorosien diasetaat gemerk en die fluoresensie gemeet deur vloeisitometrie. Insulien afskeiding deur die pankreatiese β-selle is deur radio-immuno metode bepaal. Die anti-oksidatiewe effek van die ekstrak op lipopolisakkaried-gestimuleerde perifere mononuklêere-selle afkomstig van Wistar rotte is bepaal deur die meting van TNF-α produksie met 'n ELISA kit. Resultate: C2C12 miosiete het 'n maksimale toename in glukoseopname by 'n 0.05 μg/μl ekstrakkonsentrasie (228.3% ± 66.2, p<0.001) gehad. Dieselfde ekstrakkonsentrasie het maksimale toename in glukoseopname in Chang selle (134.5% ± 2.5, p<0.05 getoon. In 3T3-L1 selle is maksimale toename in die glukoseopname by 'n konsentrasie van 0.025 μg/μl (143.5% ± 10.3, p<0.001) bereik. 'n Ekstrak-geinduseerde verhoging in die insulienreseptor en glukosetransporter vier ekspressie is in C2C12 miosiete waargeneem. Die oksidasie van 14C-glukose na 14CO2 deur C2C12 miosiete is maksimaal verhoog deur akute blootstelling aan die ekstrak by 'n konsentrasie van 0.1 μg/μl (2919.3 fmol/1x10^6 cells ± 428, p<0.01). Die oksidasie van 14C-glukose na 14CO2 deur Chang selle was maksimaal verhoog deur akute blootstelling aan die ekstrak by 'n konsentrasie van 0.1 μg/μl (4476.7 fmol/1x10^6 cells ± 1620, p<0.05) soos gevind in die C2C12 selle. Die ekstrak het glukogeenstoring verhoog teen al drie die konsentrasies waarteen getoets is in Chang selle, maar 'n maksimale effek is gevind by 'n konsentrasie van 0.025 (13.6 μg/1x10^6 cells ± 0.7, p<0.05). A. phylicoides ekstrak het nie 'n meetbare effek op die oksidatiewe status van β-selle of die anti-inflammatoriese status van perifere mononuklêere-selle gehad nie. Die ekstrak het wel 'n verhoging in die eerstefase insuliensekresie van β-selle in hyperglukemiese kondisies gehad, alhoewel die verhoging nie statisties betekenisvol was nie. Afleiding: Athrixia phylicoides waterekstrak stimuleer in vitro glukoseopname en metabolisme in 'n insulin-mimetiese manier, wat beteken dat die ekstrak potensiëel voordele vir tipe twee diabete kan inhou as aanvullingsterapie.