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The aims of this thesis were to determine the relative contribution of Pdx-1, MafA and β2 to the human insulin promoter.  Additionally, characterisation of the conserved CRE element (CRE2) was undertaken to identify the factor(s) that bind CRE2 to mediate affects upon the transcription of insulin.  Finally, the ectopic expression of insulin was investigated using an engineered zinc finger protein (ZFP-INS) that ‘switched on’ the endogenous insulin gene in a non-β cell (HEK293). Consistent with previous findings, Pdx-1, MafA and β2 were shown to synergistically activate the rat insulin 1 promoter.  However due to subtle sequence divergence, similar synergistic interaction was not observed on the human promoter.  Synergistic interactions were re-established in a rat insulin 1-like mutated human insulin construct.  The CRE binding protein activating transcription factor-2 (ATF-2) was shown to bind and stimulate transcription via CRE2 while CRE binding protein-1 (CREB-1) inhibited insulin transcription independently of CRE1 or CRE2.  ZFP-INS was shown to induce the ectopic expression of insulin in HEK293 cells via the ILPR region, inducing modifications of histone proteins at the insulin promoter.  Collectively, the continued characterisation of the human insulin promoter may reveal unique regulatory mechanisms controlling the expression of insulin under normal and diabetic conditions.

No description available.

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M.Tech. (Homeopathy) / Insulin resistance (IR) is a metabolic derangement and a documented clinical feature of the metabolic syndrome. It is an important risk factor in the development of cardiovascular disease and Type 2 Diabetes mellitus. Insulin resistance is often characterized by an increased Homeostasis Model Assessment (HOMA) index and hyperinsulinaemia, but it may also be present without increased insulin levels. Metabolic syndrome is a cluster of risk factors characterized by visceral adiposity (a girth exceeding 102cm in men and 88cm in women), dyslipidaemia (low HDL and raised triglycerides levels), hypertension and dysglycaemia, particularly raised fasting blood glucose levels, predisposing individuals to cardiovascular disease and Type 2 Diabetes mellitus. Diaglukon™ Dr Reckeweg R40 is formulated as an adjunct in the treatment of type 2 diabetes mellitus to assist in lowering the blood glucose (hyperglycaemia). The aim of the research was to evaluate and document its efficacy in the treatment of insulin resistance. A cohort of forty five participants between the ages of nineteen to forty five years was randomized into a double blind placebo controlled, 16 week, clinical study. Participants were matched according to age, race and gender. Anthropometric evaluation consisted of weight, height, BMI, waist circumference and blood pressure readings; these were recorded at 4 weekly intervals for sixteen weeks. Metabolic data included fasting insulin, glucose and a full lipogram at baseline (Week 0) and at study conclusion (Week 16). The insulin and glucose was used to calculate the HOMA index as a measure of insulin resistance (IR). Non parametric statistical analysis was conducted on all parameters using the SPSS statistical programme...

The insulin receptor ortholog EmIR of the fox-tapeworm Echinococcus multilocularis displays significant structural homology to the human insulin receptor (HIR) and has been suggested to be involved in insulin sensing mechanisms of the parasite’s metacestode larval stage. In the present work, the effects of host insulin on Echinococcus metacestode vesicles and the proposed interaction between EmIR and mammalian insulin have been studied using biochemical and cell-biological approaches. Human insulin, exogenously added to in vitro cultivated parasite larvae, (i) significantly stimulated parasite survival and growth, (ii) induced DNA de novo synthesis in Echinococcus, (iii) affected overall protein phosphorylation in the parasite, and (iv) specifically induced the phosphorylation of the parasite’s Erk-like MAP kinase orthologue EmMPK1. These results clearly indicated that Echinococcus metacestode vesicles are able to sense exogenous host insulin which induces a mitogenic response. To investigate whether EmIR mediates these effects, anti-EmIR antibodies were produced and utilized in biochemical assays and immunohistochemical analyses. EmIR was shown to be expressed in the germinal layer of the parasite both on the surface of glycogen storing cells and undifferentiated germinal cells. Upon addition of exogenous insulin to metacestode vesicles, the phosphorylation of EmIR was significantly induced, an effect which was suppressed in the presence of specific inhibitors of insulin receptor-like tyrosine kinases. Furthermore, upon expression of EmIR/HIR receptor chimera containing the extracellular ligand binding domain of EmIR in HEK 293 cells, a specific autophosphorylation of the chimera could be induced through the addition of exogenous insulin. These results indicated the capability of EmIR to sense and to transmit host insulin signals to the Echinococcus signaling machinery. The importance of insulin signaling mechanisms for parasite survival and growth were underscored by in vitro cultivation experiments in which the addition of an inhibitor of insulin receptor tyrosine kinases led to vesicle degradation and death. Based on the above outlined molecular data on the interaction between EmIR and mammalian insulin, the parasite’s insulin receptor orthologue most probably mediates the insulin effects on parasite growth and is, therefore, a potential candidate factor for host-parasite communication via evolutionary conserved pathways. In a final set of experiments, signaling mechanisms that act downstream of EmIR have been analyzed. These studies revealed significant differences between insulin signaling in Echinococcus and the related cestode parasite Taenia solium. These differences could be associated with differences in the organo-tropism of both species. / Der orthologe Insulinrezeptor EmIR des Fuchsbandwurmes Echinococcus multilocularis weist signifikante strukturelle Homologie zum humanen Insulinrezeptor (HIR) auf. Es wurde schon seit geraumer Zeit vermutet, dass EmIR an den Mechanismen beteiligt sein könnte, die es dem Metacestoden Larvenstadium des Parasiten erlauben Insulin zu detektieren. In dieser Arbeit wurden die Effekte von Wirtsinsulin auf Echinococcus Metacestoden-Vesikel und die vermutete Interaktion zwischen EmIR und Insulin von Säugern mittels biochemischer und zellbiologischer experimenteller Ansätze untersucht. Die exogene Zugabe von humanem Insulin zu in vitro kultivierten Parasitenlarven hatte folgende Effekte: (i) das Überleben und das Wachstum des Parasiten wurde signifikant stimuliert; (ii) die DNA de novo Synthese in Echinococcus wurde induziert; (iii) die generelle Proteinphosphorylierung des Parasiten wurde beeinflusst; (iv) die Phosphorylierung der orthologen Erk-like MAP Kinase, EmMPK1, des Parasiten wurde spezifisch induziert. Diese Beobachtungen zeigen deutlich, dass Echinococcus Metacestoden-Vesikel exogenes Insulin des Wirtes detektieren können und dass dieses Insulin einen mitogenischen Effekt auf den Parasiten hat. Um zu untersuchen, ob diese Effekte durch EmIR vermittelt werden, wurden anti-EmIR Antikörper hergestellt und in biochemischen experimentellen Ansätzen und immunohistochemischen Analysen eingesetzt. Es konnte gezeigt werden, dass EmIR in der Germinalschicht des Parasiten expremiert wird, sowohl an der Oberfläche von Glykogen-Speicherzellen als auch von undifferenzierten Germinalzellen. Nach der Zugabe von exogenem Insulin konnte eine signifikante Zunahme der Phosphorylierung von EmIR festgestellt werden. Diese Stimulierung konnte durch die Zugabe eines spezifischen Inhibitors für Insulinrezeptor-ähnliche Tyrosinkinasen unterdrückt werden. Desweiteren konnte mittels der Expression eines chimären EmIR/HIR-Rezeptors, der die extrazelluläre Ligandenbindungsdomäne von EmIR enthielt, in HEK293 Zellen gezeigt werden, dass die Zugabe von exogenem Insulin eine spezifische Autophosphorylierung der Chimäre induziert. Diese Ergebnisse bezeugen die Fähigkeit von EmIR Insulin-abhängige Signale des Wirtes einerseits zu detektieren und andererseits an die Echinococcus Signalwege weiter zu leiten. Die Bedeutung von Insulin-Signalmechanismen für das Überleben und das Wachstum des Parasiten konnte durch in vitro Kultivierungsexperimente aufgezeigt werden. Die Zugabe eines Inhibitors spezifisch für Insulinrezeptor Tyrosinkinasen verursachte die Degradation und den Tod der Metacestoden-Vesikel. Basierend auf den dargelegten molekularen Daten bezüglich der Interaktion zwischen EmIR und Insulin von Säugern erscheint es sehr wahrscheinlich, dass der orthologe Insulinrezeptor des Parasiten die Effekte von Insulin auf das Wachstum des Parasiten vermittelt. Aus diesem Grund ist EmIR ein potentieller Kandidat für die Kommunikation zwischen Wirt und Parasiten mittels evolutionär konservierten Signalwegen. Die Signalmechanismen unterhalb von EmIR wurden in abschließenden Experimenten untersucht. Diese offenbarten deutliche Unterschiede in der Weiterleitung von Insulin induzierten Signalen zwischen Echinococcus und dem verwandten parasitären Zestoden Taenia solium. Diese Unterschiede könnten mit dem unterschiedlichen Organtropismus beider Arten in Verbindung stehen.

Monozyten lassen sich in vitro nicht nur zu Makrophagen und Dendritischen Zellen differenzieren, sondern auch in eine Vielzahl nicht-phagozytierender Zellen. Monozyten scheinen somit über pluripotente Eigenschaften zu verfügen. In dieser Arbeit wurde untersucht, ob sich kultivierte Monozyten tatsächlich in Insulin-exprimierende Zellen differenzieren lassen. Monozyten von gesunden Spendern im Alter zwischen 20 und 26 Jahren wurden untersucht. Die über eine Leukozytenapherese gewonnenen Mono- zyten wurden über Adhärenz angereichert und für sechs Tage in X-Medium mit den Cytokinen M-CSF und IL-3 und für weitere 15 Tage in Y-Medium mit den Cytokinen HGF und EGF inkubiert. Die Zellen wurden immunhistochemisch und funktionell untersucht. Frisch isolierte Blutmonozyten waren vor ihrer Kultivierung negativ für Insulin, C-Peptid und Glukagon. Am 4. Kulturtag wurden Insulin und C-Peptid in den kultivierten Monozyten nachgewiesen. Die Expression von Insulin war jedoch nicht stabil: während am Tag 11 der Anteil Insulin-positiver Zellen bei ca. 80% lag, waren am Tag 14 nur noch ca. 30% der kultivierten Zellen Insulin-positiv. Dies wurde ebenfalls für den Nachweis von C-Peptid beobachtet. Auch die Expression von Glukagon war nicht stabil. Diese Beobachtung wird darauf zurückgeführt, dass sich die Monozyten zu Makrophagen differenzierten und diese eindeutig kein Insulin produzieren. Da Zellen Insulin aufnehmen und speichern können, sollte in dieser Arbeit die Frage geklärt werden, ob immunhistochemisch zu unterscheiden ist, ob Zellen Insulin gebildet (de novo Insulin) oder unspezifisch aufgenommen haben. In dieser Arbeit wurde gezeigt, dass zum eindeutigen Nachweis von de novo Insulin der Nachweis von C-Peptid unbedingt zu fordern ist. Die in dieser Arbeit durchgeführten Experimente mit aufgereinigtem Insulin belegen, dass für aufgenommenes Insulin – im Gegensatz zu de novo Insulin – C-Peptid immun- histochemisch nicht nachzuweisen ist. Das aus in vitro kultivierten Monozyten isolierte Insulin war in diabetischen Mäusen biologisch aktiv, d.h. es senkte den Blutzuckerspiegel kurzfristig. Hierzu wurden geerntete Monozyten der Kulturtage 6-12 im Ultraschallbad aufgeschlossen und der zellfreie Überstand diabetischen Mäusen injiziert. Insgesamt senkten 11 der 31 (35,5%) in dieser Arbeit getesteten Überstände den Blutzuckerspiegel dieser Tiere um mehr als 15%. Bezogen auf die 18 Proben, die einen Effekt zeigten, sind dies sogar 61%. In dieser Arbeit wurde somit erfolgreich gezeigt, dass in vitro modifizierte Monozyten Insulin exprimieren, das den Blutzuckerspiegel diabetischer Mäuse senkt. / Monocytes differentiate not only in macrophages and dendritic cells but also in a variety of non-phagocytic cells. Monocytes or a subpopulation of monocytes seem to exhibit pluripotent diversity. In the present study the potential of in vitro cultured monocytes to differentiate into insulin-expressing cells was analysed. Monocytes of healthy human donors between 20 and 26 years old were analysed. They were obtained by leukocyte aphaeresis, enriched by adherence and cultured six days in X-Medium containing MCS-F und IL-3 and a further 15 days in Y-Medium containing HGF and EGF. The cells were characterized by immunohistochemistry and functional assays. Freshly isolated blood monocytes were negative for insulin, C-peptide and glucagon. In contrast, cultured monocytes were positive for insulin and C-peptide, detectable after 4 days in culture. However, the insulin expression was unstable. On day 11 of culture the amount of insulin-positive cells reached a maximum of 80 percent positive, by day 14 of culture the amount of insulin-positive cells had decreased to 30 percent. Similar results were obtained for C-peptide and glucagon. It seems that monocytes lost the ability to express insulin during their differentiation into macrophages. It is well known, that cells are able to take up and accumulate exogenous insulin. One aim of this study was to differentiate by immunohistochemistry between de novo insulin and exogenous insulin taken up by the cells from the environment. We showed that the presence of C-peptide must be proven to clearly identify de novo insulin. Experiments with purified insulin demonstrated that cells that took up purified insulin were negative for C-peptide. Insulin isolated from in vitro cultured monocytes demonstrated biological activity and reduced blood glucose levels in diabetic mice. Eleven of 31 in vivo tested cell-free supernatants (35.5 percent) lowered blood glucose levels in diabetic mice by more than 15 percent. The results of the study show that in vitro modified monocytes are able to produce insulin that reduces blood glucose levels in diabetic mice.

MMed, Paediatrics, University of the Witwatersrand, Faculty of Health Sciences / Background: To reduce the risks and prevent progression of diabetic complications average blood glucose and glucose variability need to be kept as close to the non diabetic range as possible. Post prandial glucose excursions contribute significantly to average blood glucose and to glycemic variability. Dietary carbohydrate is the primary determinant of meal related blood glucose excursions. Carbohydrate counting is a method of insulin dosing that matches carbohydrate load to insulin dose using a fixed ratio. Many patients and current insulin pumps, calculate insulin delivery for meals based upon a linear carbohydrate to insulin relationship. Hypothesis: A non-linear relationship exists between the amount of carbohydrate consumed and the insulin required to cover it. Rather, an exponential increase in insulin is needed to cover an increasing load of carbohydrate. Aim: To document blood glucose exposure, as measured by AUC, in response to increasing carbohydrate loads on fixed carbohydrate to insulin ratios. Sample and Methods: 5 Type-1 diabetic adolescents and young adults on insulin pump therapy with good control were recruited. Morning basal rates and carbohydrate to insulin ratios were optimized prior to the study start. A Medtronic glucose sensor was worn by each participant for 5 days on which standardized meals of increasing carbohydrate content were consumed. After the 5 days the glucose sensors were downloaded and the glucose area under the curve was analyzed for each carbohydrate load for each participant. Results: Only subjects with 5 days of complete recordings covering the test meals were included for analysis, resulting in 5 complete analyses. Sensor failure and hypoglycaemic v episodes prior to test meals accounted for failures. Increasing carbohydrate loads on a fixed carbohydrate to insulin ratio resulted in increasing glucose area under the curve (AUC).The log (Average AUC) was linear confirming that this relationship is exponential. An Analysis of Covariance performed on the log (AUC) data confirmed a highly significant exponential relationship (p<0.0001) although no significant differences were found between the profiles of the 5 individuals. Late post prandial hypoglycaemia followed carbohydrate loads greater than 60 grams and this was often followed by rebound hyperglycaemia that lasted more than 6 hours. Conclusion: A non linear relationship exists between carbohydrates consumed and the insulin required to cover them when using premeal bolus insulin. This has implications for control of postprandial blood sugars, especially when consuming large carbohydrate loads. Because of the late post prandial hypoglycaemia that follows the larger doses of insulin used with larger amounts of carbohydrate it is not possible to simply increase the amount of the insulin bolus using an exponential formula. Further studies need to be done looking at the optimal ratios of insulin needed for increasing carbohydrate loads, the duration and type of boluses needed to cover these high carbohydrate loads and the possibility of changing the linear equation used in current insulin pumps to one that would better cover the increase in post prandial glucose load with large carbohydrate meals.

MSc Pharmacology, Faculty of Health Sciences, University of the Witwatersrand / Natural remedies from medicinal plants are considered to be effective and safe alternative treatment for diabetes mellitus. The aim of this study was to demonstrate the hypoglycaemic and antidiabetic activity of the aqueous extract of Icacina tracantha (tuber) (fam Icacinaceae)Ananas cosmos (fam. Bromeliaceae)and Uraria picta (leaves) (fam leguminosae) on an animal model of insulin resistance, a condition which predisposes to type 2 diabetes. The plants have a long history of use as anti-diabetic agents in western Nigeria. Method: 120 male Sprague-Dawley rats were assigned into two major groups. One group was fed on normal rat chow with the other group fed on a high calorie diet for four months a period sufficient for the animals to be fed to attain insulin resistance. The animals were then randomly assigned into different groups (each containing 6 male rats). The plant crude extracts were made by weighing specific dried quantities of each plant, boiling in distilled water for about 2 hours, cooling overnight and separating solid from liquid by filtration. The solution was then poured into preweighed 250 ml beakers and allowed to dry in an oven at a temperature of 60oC. The dried, crude extracts were then weighed out and required doses prepared from the extracts. A non-treated group of animals was used as the control. The mixed dose of extract was administered at 300 mg/kg. Over a 3 week period, all the animals were orally dosed with the different doses of plant extracts daily while metformin was administered through the animals’ drinking water, blood was collected from the tail vein of each rat prior to dosing and thereafter weekly, plasma was preserved and 6 analysed for glucose, insulin, free fatty acid concentrations and calculation of HOMA values to determine insulin sensitivity. During this period, the animals were weighed weekly and food intake was measured every three days. An oral glucose tolerance test (OGTT) was performed after the dosing period and fasting, 0, 30, 60 and 120 minute blood samples were taken and assayed for glucose concentration. Animals were terminated and blood analysed. Statistical analysis: The results were tabulated as mean ± standard deviation and percentage median ± quartile range. The statistical analysis for other parameters was carried out via ANOVA (between groups) and Student’s paired T test (within groups). Only data from percentage median and quartile range was used because of the observed variation in glucose concentration between groups even at baseline values. Statistica software (StatSoft, Tulsa, OK, USA) was used for the analysis. Results: All plant extracts in the study showed differing concentration of significant difference in their effect on the plasma glucose, insulin and free fatty acid concentrations in the rat. The most significant effect was observed on the insulin concentration in the normal rat chow and high calorie diet fed animals. The plant extracts were observed to improve insulin sensitivity in most of the groups. This effect was more significant in the normal rat chow fed rats. The effect of the plant extracts on the weight, food consumed glucose and free fatty acid was minimal and in most of the groups was not significant. Conclusion: In conclusion, the results obtained suggest that the plant extracts may be used to improve insulin resistance in the management of diabetes mellitus.