Les décès attribués à un choc septique à la suite d’une infection sévère augmentent chez les diabétiques et surviennent assez fréquemment dans les unités de soins intensifs. Le diabète sucré et le choc septique augmentent la production d’espèces réactives oxygénées et de cytokines pro-inflammatoires, lesquelles activent le facteur de transcription nucléaire Kappa B conduisant à l’induction du récepteur B1 (RB1) des kinines. Le diabète induit par la streptozotocine (STZ) augmente l’expression du RB1 dans divers tissus périphériques, le cerveau et la moelle épinière. Les lipopolysaccharides bactériens (LPS), souvent utilisés pour induire le choc septique, induisent aussi le RB1. L’objectif de ce travail vise à démontrer la contribution du RB1 des kinines dans l’exacerbation du choc septique pendant le diabète. Des rats Sprague-Dawley (225-250 gr) traités à la STZ (65 mg/kg, i.p.) ou le véhicule ont reçu quatre jours plus tard les LPS (2 mg/kg, i.v.) ou le véhicule en présence ou pas d’un antagoniste du RB1 (SSR240612, 10 mg/kg) administré par gavage. La température corporelle a été mesurée pendant 24h après le traitement. Le SSR240612 a aussi été administré à 9h AM et 9h PM et les rats sacrifiés à 9h AM le jour suivant après un jeûne de 16 h. Les effets de ces traitements ont été mesurés sur les taux plasmatiques d’insuline et de glucose, l’œdème et la perméabilité vasculaire (dans divers tissus avec la technique du Bleu d’Evans) ainsi que sur l’expression du RB1 (PCR en temps réel) dans le cœur et le rein. L’augmentation de la température corporelle après traitement au LPS chez les rats traités ou pas à la STZ a été bloquée par le SSR240612. L’antagoniste a normalisé l’hyperglycémie et amélioré la déficience en insuline chez les rats STZ. Le SSR240612 a inhibé l’œdème et réduit la perméabilité vasculaire dans les tissus des rats diabétiques traités ou pas avec les LPS. La surexpression du RB1 chez les rats traités au STZ et/ou LPS était renversée par le SSR240612. Cet antagoniste a prévenu la mortalité causée par les LPS et LPS plus STZ. Les effets anti-pyrétique, anti-inflammatoire et anti-diabétique du SSR240612 suggèrent que le RB1 puisse représenter une cible thérapeutique valable pour le traitement de la co-morbidité associée au choc septique dans le diabète. / Death attributed to septic shock following severe infection increases in diabetic patients and occurs quite frequently in intensive care units. Diabetes mellitus and septic shock increase the production of reactive oxygen species and pro-inflammatory cytokines leading to higher kinin B1 receptor (RB1) expression that is mediated by the activation of the transcriptional nuclear factor Kappa B. Streptozotocin (STZ)-induced diabetes increased the expression of RB1 in rat peripheral tissues, brain and spinal cord. Bacterial lipopolysaccharides (LPS) commonly used to induce septic shock in animal models, also induce RB1. Our objective is to study the contribution of kinin RB1 in the increased morbidity and mortality associated with the combination of these two diseases. Sprague-Dawley rats (225-250g) treated with STZ (65 mg/kg, ip) or vehicle received four days later LPS (2 mg/kg, iv) or vehicle in the presence or absence of the RB1 antagonist, SSR240612 (10 mg/kg), administered by gavage. Body temperature was monitored for 24h after treatment. In addition, SSR240612 was administered twice (9h AM and 9h PM) and rats were sacrificed the following morning at 9h AM after 16 h of fasting to measure the impact on plasma insulin and glucose, oedema and vascular permeability in various tissues (with the technique of Evans Blue) and on the expression of RB1 (real-time PCR) in heart and kidney. The increase in body temperature caused by treatment with LPS both in STZ-diabetic and non-diabetic rats was blocked by SSR240612. The antagonist normalized hyperglycaemia and improved insulin deficiency in STZ rats. SSR240612 inhibited oedema and reduced vascular permeability in all tissues from diabetic rats treated or not with LPS. The overexpression of RB1 induced by LPS and STZ was blocked by SSR240612. Pharmacological blockade of B1R with SSR240612 prevented the mortality induced by LPS and STZ plus LPS. Thus the anti-pyretic, anti-inflammatory and anti-diabetic effects of SSR240612 suggest that kinin RB1 is a promising therapeutic target for the treatment of co-morbidity associated with septic shock in diabetes.

Diabetes mellitus currently affects over 422 million people globally and over 80% of patients with diabetes will develop diabetic retinopathy. Patients with diabetic retinopathy initially develop background retinopathy, which does not cause significant deterioration of visual function; however, background retinopathy may progress and lead to proliferative diabetic retinopathy and diabetic macular oedema, both of which cause severe visual dysfunction if left untreated. Current therapies for diabetic retinopathy include invasive intravitreal injections and laser photocoagulation; however these treatments only attenuate the progression of proliferative diabetic retinopathy and diabetic macular oedema. Aside from prevention by maintaining good blood glucose and blood pressure control, there are currently no treatments to prevent progression to late-stage diabetic retinopathy and new innovations in the field have not significantly progressed. For this reason, we have used untargeted –omics approaches to identify previously unknown pathological pathways in diabetes. In this thesis, I have analysed a range of trace metals in donor retinas and found that total copper was increased in diabetic retinas compared with non-diabetic. This result was replicated in streptozotocin-induced diabetic rat retinas and further evidenced by upregulation of metallothioneins and caeruloplasmin in diabetic rat retinas compared with non-diabetic. Treatment with the copper chelator triethylenetetramine modulated these changes, the downstream effects of which require further study. This is the first description, to our knowledge, of dysregulated copper homeostasis in the diabetic retina. I have also mapped metabolic changes in streptozotocin-induced diabetic rat retinas and found previously undescribed metabolite changes such as diabetes-induced downregulation of scyllo inositol. This coincided with substantial changes to retinal lipids during diabetes and changes to individual lipids were consistent within their respective class. I have also found a pattern whereby regardless of the extent of change to a lipid class in diabetes, lipids containing docosahexaenoic acid (22:6 carbon chain) were consistently downregulated. This is thought to be the first study to describe this range of metabolite changes in the diabetic retina but also the first study to describe this range of metabolite analysis concomitantly within the same tissue sample. The data from this study provides new insights into metallomic and metabolic dysfunction in diabetic retinopathy and shown that these data are reproducible. We suggest that there is plenty of scope for further research to investigate mechanisms behind copper dysregulation, how this affects pathogenesis of diabetic retinopathy along with new insights into dysregulated metabolic pathways.

No description available.

Thesis submitted in fulfillment of the requirements for the Doctor of Technology: Biomedical Technology In the Faculty of Health and Wellness At the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY 2014 / Diabetes mellitus (DM) results in severe metabolic imbalances and pathological changes in many tissues. Chronic inflammation and oxidative stress have been implicated in the pathophysiology of diabetes mellitus. Garcinia kola (Family: Guttiferae) is a plant well known for its ample medicinal values. The seed of the plant also known as ‘bitter kola’ due to its bitter taste is used as a masticatory agent in traditional hospitality, cultural and social ceremonies in Africa. Kolaviron (KV) is a defatted ethanol extract from the seeds of Garcinia kola (GK). Kolaviron has been shown in experimental models of diseases to have numerous beneficial effects due to the presence of flavonoids (mainly Garcinia biflavonoid (GB)-1, GB-2 and kolaflavanone). However, there is paucity of information regarding the possible effect of kolaviron on inflammatory mediators and oxidative stress in diabetes mellitus. Therefore, this study was carried out to investigate the potential beneficial effects of kolaviron on antioxidant status, inflammatory mediators and apoptosis. Other biochemical and histological alterations in the blood, liver and kidney of streptozotocin-induced diabetic rats were also evaluated. A single intraperitoneal injection of freshly prepared solution of streptozotocin (50 mg/kg.b.wt.) in citrate buffer (0.1M, pH 4.5) was administered to overnight fasted rats for diabetes induction. Diabetes was confirmed by stable hyperglycemia (>18 mmol/l) in the tail blood glucose after 5 days of streptozotocin injection. Kolaviron (100 mg/kg b.wt.) was administered to diabetic rats (by gastric gavage) on the 6th day after the induction of diabetes and treatment continued for 6 weeks (5 times weekly). The effects on blood glucose, body weight, organ (liver and kidney) weight, serum biochemical parameters, oxidative status, inflammatory mediators and histology of the liver, kidney and pancreas were assessed. Kolaviron (KV) treatment lowered blood glucose in diabetic and normoglycemic rats and reduced glycated haemoglobin [HbA1C (%)]. Plasma insulin level was raised in diabetic rats treated with KV. Histomorphometric analysis of the pancreas revealed increased β-cell area of pancreatic islets of kolaviron-treated diabetic group. The indices of organ (liver and kidney) damage were increased in diabetic rats. However, KV treatment protected against liver and kidney damage. The characteristic features of diabetic dyslipidemia such as elevated serum triglyceride and cholesterol concentration which are major risk factors for cardiovascular disease were also significantly reduced in KV-treated diabetic rats. Alteration in antioxidant enzymes status was observed in the liver, kidney and blood (erythrocyte, plasma and serum) of diabetic rats. Lowered catalase (CAT) activity was observed in the liver and kidney of diabetic rats while KV treatment significantly (p < 0.05) elevated catalase activity in the liver and kidney. There was no significant change (p > 0.05) in erythrocyte catalase activity among all treatment groups. Erythrocyte of diabetic rats showed a marked reduction in the activity of superoxide dismutase (SOD) with no significant changes in liver and kidney SOD activity of diabetic rats compared to control whereas KV administration to rats markedly increased SOD activity. Glutathione peroxidase (GPX) activity was elevated in the erythrocyte and kidney of STZ-induced diabetic rats with no significant effect on liver GPX activity. KV treatment reversed the alteration in GPX activity in the kidney and erythrocyte. Level of reduced glutathione (GSH), a non-enzymatic antioxidant was decreased in the both liver and kidney of diabetic rats and treatment of diabetic rats with KV elevated GSH concentration in both tissues. Also, malondialdehyde (MDA), a marker of lipid peroxidation was elevated in the liver, kidney and plasma of diabetic rats and significantly (p < 0.05) lowered following KV treatment. Diabetes induction reduced the capacity of liver and kidney to absorb oxygen radicals as demonstrated by lowered oxygen radical absorbance capacity (ORAC) values. KV administration to normal and diabetic rats significantly increased ORAC values. Increased rate of apoptosis, a major cellular response to high glucose induced stress was observed in the renal and hepatic tissues of diabetic control rats. Kolaviron treatment of diabetic rats protected the liver and kidney against hyperglycemia-induced apoptosis and decreased the number of TUNEL positive cells A significant (p < 0.05) elevation of pro-inflammatory cytokines; monocyte chemoattractant protein (MCP-1), Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor (TNF)-𝛂 was observed in the liver of diabetes rats. KV treatment lowered these inflammatory biomarkers. On the other hand, the kidney of diabetic rats showed elevated concentration of pro-inflammatory IL-1β with no significant effect on kidney TNF-𝛂. An increase in the serum concentration of MCP-1 and IL-1β was observed in the untreated diabetic rats while kolaviron treatment normalized the alteration in serum concentration of MCP-1, IL-1β and vascular endothelial growth factor (VEGF). In conclusion, persistent and chronic hyperglycemia promotes the generation of free radicals and inflammatory molecules which contributes to progressive development of micro- and macro vascular complications and multi-organ damage. Kolaviron demonstrated beneficial effects on markers of oxidative stress and inflammation in the diabetic rats and also promoted the survival and functional integrity of the liver and kidney.

Indiana University-Purdue University Indianapolis (IUPUI) / Intestinal smooth muscle development involves complex transcriptional regulation leading to cell differentiation of the circular, longitudinal and muscularis mucosae layers. Differentiated intestinal smooth muscle cells express high levels of smooth muscle-specific contractile and regulatory proteins, including telokin. Telokin is regulatory protein that is highly expressed in visceral smooth muscle. Analysis of cis-elements required for transcriptional regulation of the telokin promoter by using hypoxanthine-guanine phosphoribosyltransferase (Hprt)-targeted reporter transgenes revealed that a 10 base pair large CC(AT)₆GG ciselement, called CArG box is required for promoter activity in all tissues. We also determined that an additional 100 base pair region is necessary for transgene activity in intestinal smooth muscle cells. To examine how transcriptional regulation of intestinal smooth muscle may be altered under pathological conditions we examined the effects of diabetes on colonic smooth muscle. Approximately 76% of diabetic patients develop gastrointestinal (GI) symptoms such as constipation due to intestinal dysmotility. Mice were treated with low-dose streptozotocin to induce a type 1 diabetes-like hyperglycemia. CT scans revealed decreased overall GI tract motility after 7 weeks of hyperglycemia. Acute (1 week) and chronic (7 weeks) diabetic mice also had decreased potassium chloride (KCl)-induced colon smooth muscle contractility. We hypothesized that decreased smooth muscle contractility at least in part, was due to alteration of contractile protein gene expression. However, diabetic mice showed no changes in mRNA or protein levels of smooth muscle contractile proteins. We determined that the decreased colonic contractility was associated with an attenuated intracellular calcium increase, as measured by ratio-metric imaging of Fura-2 fluorescence in isolated colonic smooth muscle strips. This attenuated calcium increase resulted in decreased myosin light chain phosphorylation, thus explaining the decreased contractility of the colon. Chronic diabetes was also associated with increased basal calcium levels. Western blotting and quantitative real time polymerase chain reaction (qRT-PCR) analysis revealed significant changes in calcium handling proteins in chronic diabetes that were not seen in the acute state.These changes most likely reflect compensatory mechanisms activated by the initial impaired calcium response. Overall my results suggest that type 1 diabetes in mice leads to decreased colon motility in part due to altered calcium handling without altering contractile protein expression.

Several studies suggest that diabetes affects male and female vascular beds differently. However, the mechanisms underlying the interaction of sex and diabetes remain to be investigated. This study investigates whether there are 1) sex differences in the development of abnormal vascular responses and 2) changes in the relative contributions of endothelium-derived relaxing factors (EDRFs) in modulating vascular reactivity of mesenteric arteries taken from streptozotocin (STZ)-induced diabetic rats at early and intermediate stages of the disease (one and eight weeks, respectively). We also investigated the mesenteric expression of the mRNAs for endothelial nitric oxide synthase (eNOS) and NADPH oxidase (Nox) in STZ-induced diabetes in both sexes. Vascular responses to acetylcholine (ACh) in mesenteric arterial rings pre-contracted with phenylephrine were measured before and after pretreatment with indomethacin (cyclooxygenase inhibitor), L -NAME (NOS inhibitor), or barium chloride (K IR blocker) plus ouabain (Na + -K + ATPase inhibitor). We demonstrated that ACh-induced relaxations were significantly impaired in mesenteric arteries from both male and female diabetic rats at one and eight weeks. However, at eight weeks the extent of impairment was significantly greater in diabetic females than diabetic males. Our data also showed that in females, the levels of eNOS and Nox2- and Nox4-dependent NADPH oxidase mRNA expression and the relative importance of NO to the regulation of vascular reactivity were substantially enhanced, while the importance of endothelium-derived hyperpolarizing factor (EDHF) was significantly reduced at both one and eight weeks after the induction of diabetes. This study reveals the predisposition of female rat mesenteric arteries to vascular injury after the induction of diabetes, may be due to a shift away from a putative EDHF, initially the major vasodilatory factor, towards a greater reliance on NO, and the interaction of oxidative stress with elevated NO.