Diagnosis of diabetes mellitus, presence of hyperglycemia, and/or insulin resistance confer cardiovascular risk, particularly for diastolic dysfunction. Diabetes is associated with elevated myocardial norepinephrine (NE) content, enhanced sympathetic nervous system (SNS) activity, altered resting heart rate, and depressed heart rate variability. Positron emission tomography (PET) using the NE analogue [11C]meta-hydroxyephedrine ([11C]HED) provides an index of myocardial sympathetic neuronal integrity at the NE reuptake transporter (NET). The hypothesis of this project is that (i) hyperglycemia imparts heightened sympathetic tone and NE release, leading to abnormal sympathetic neuronal function in the hearts of diabetic rats, and (ii) these abnormalities may be reversed or prevented by treatments to normalize glycemia. Sprague Dawley rats were rendered insulin resistant by high fat feeding and diabetic by a single dose of streptozotocin (STZ). Diabetic rats were treated for 8 weeks with insulin, metformin or rosiglitazone, starting from either 1 week (prevention) or 8 weeks (reversal) after STZ administration. Sympathetic neuronal integrity was evaluated longitudinally by [11C]HED PET. Echocardiography measures of systolic and diastolic function were completed at serial timepoints. Plasma NE levels were evaluated serially and expression of NET and β-adrenoceptors were tested at the terminal endpoints. Diabetic rats exhibited a 52-57% reduction of [11C]HED standardized uptake value (SUV) at 8 weeks after STZ, with a parallel 2.5-fold elevation of plasma NE and a 17-20% reduction in cardiac NET expression. These findings were confirmed by ex vivo biodistribution studies. Transmitral pulse wave Doppler echocardiography established an extension of mitral valve deceleration time and elevated early to atrial velocity ratio, suggesting diastolic dysfunction. Subsequent treatment with insulin but not metformin restored glycemia, reduced plasma NE by 50%, normalized NET expression, and recovered [11C]HED SUV towards non-diabetic age-matched control. Diastolic dysfunction in these rats persisted. By contrast, early treatment with insulin, metformin, or rosiglitazone delayed the progression of diastolic dysfunction, but had no effect on elevated NE and reduced [11C]HED SUV in diabetic rats, potentially owing to a latent decrease in blood glucose. In conclusion, diabetes is associated with heightened circulating and tissue NE levels which can be effectively reversed by lowering glycemia with insulin. Noninvasive interrogation of sympathetic neuronal integrity using [11C]HED PET may have added value in the stratification of cardiovascular risk among diabetic patients and in determining the myocardial effects of glycemic therapy.

There are many tools available for the study of post-translational modifications. The majority of these tools is specific towards the individual modification and involves separation of modified proteins from non-modified ones. The drawback of using a modification specific method is that there is a lack of flexibility in its usage for other modifications. The goal of these studies was to investigate the possibility of obtaining a similar separation effect by fractionating post-translationally modified proteins based on the physical properties of proteins. The post-translational modification chosen to be the basis of this study was the O-GlcNAc modification. Using the C2C12 mouse myoblast cell line, it was determined that the optimal conditions for producing lysates containing increased yields of O-GlcNAc modified proteins was to treat differentiated C2C12 cells with 10nM insulin, 12g/L glucose and 2mM of the O-GlcNAcase inhibitor Streptozotocin for 24 hours. Using the optimized lysis buffer, it was shown that protein separation by surface charge using standard anion exchange separation did not provide enough resolution or material to obtain any identifications of modified proteins. However, when a chromatofocusing method which separates proteins on the basis of their isoelectric points was used, a separation scheme with larger capacity and higher resolution was possible. Using this separation method followed by gel electrophoresis of individual fractions, proteins which are potentially O-GlcNAc modified were identified by mass spectrometry. It was evident from the number of protein bands observed per fraction on the Coomassie stained gels and the number of proteins identified per protein band by mass spectrometry that further reduction in sample complexity was required to assist in the positive identification of O-GlcNAc modified proteins. Among the identified proteins, 32 percent were metabolic proteins, 21 percent were protein processing proteins, 16 percent were structural proteins and the remainder a mix of other proteins. Unfortunately, it was not possible to validate the presence or absence of the O-GlcNAc modification on these proteins using available methodologies such as immunoprecipitation. As such, further work is required to optimize the separation strategy and to verify the usefulness of this separation strategy in identifying O-GlcNAc/post-translationally modified proteins.

This study was prompted by the inconsistent reports and apparent controversies that exist in the biomedical literature on the responses of diabetic bladder strips to cholinergic nerve stimulation or exogenous administration of muscarinic agonists, especially acetylcholine (ACh), in vitro. In the present study, acetylcholine-induced contractions of urinary bladders isolated from normoglycaemic (normal) and streptozotocin-treated, diabetic Wistar rats were examined under physiological conditions. Mechanical contractile changes of the isolated urinary bladders of STZ-treated, diabetic rats in response to bath-applied acetylcholine were compared with those obtained from isolated urinary bladders of normal, age-matched, control rats. Results obtained show that urinary bladders from diabetic rats consistently weighed more, and were always more spontaneously active after mounting, than those of the age-matched normal, control rats. ft A Acetylcholine (ACh, 10" -10" M) provoked concentration-related, atropine-sensitive contractions of the isolated urinary bladders of both diabetic and age-matched normal, control rats. However, acetylcholine always induced more powerful and greater contractions of the diabetic bladders compared with bladders from the age-matched normal, control rats. The enhanced contractile responses of the diabetic bladder strips to bath-applied ACh were detected soon after induction of diabetes, and the magnitude and/or intensity of the enhanced contractile responses to ACh continued to increase as the diabetic state of the animals progressed. Although this preliminary study could not establish the mechanism of the increased contractile responsiveness of the diabetic bladders to the muscarinic agonist (ACh) used, the results tend to suggest that alterations in diabetic urinary bladder synaptosomal, vesicle-bound neurotransmitter (ACh) concentrations and the compensatory increase in the density of muscarinic M3-receptor population in diabetic bladders are two of the most attractive plausible mechanisms of the increased diabetic bladder responsiveness to bath-applied acetylcholine. / Thesis (M.Sc.Pharm.)-University of KwaZulu-Natal, Westville, 2006.

Treatment of diabetes mellitus is mainly focused on glycaemic control regulated by insulin and takes place in insulin sensitive tissues like skeletal muscle which accounts for 75% of glucose metabolism. Plant derived compounds that have anti-diabetic potential are currently being investigated for diabetes treatment as they are cheap and non-toxic. Oleanolic acid (OA), a triterpene found in a wide variety of plants has been shown to have anti-diabetic effects but its mechanism of action, especially on the insulin signalling cascade has not been fully elucidated. The aim of the present study was to investigate the effects of OA on the PI3K/Akt insulin signalling cascade in skeletal muscle and skin of streptozotocin induced diabetic male Sprague-Dawley rats. Male Sprague-Dawley rats (non-diabetic and diabetic) were treated with insulin (4IU/ kg bw), OA (80 mg/kg bw) and a combination of OA + insulin in an acute and sub-chronic study. The study showed that OA does not reduce blood glucose levels in type 1 diabetic rats but enhances insulin stimulated hypoglycaemic effects. In the acute study OA was shown to activate Akt and dephosphorylate GS in skeletal muscle of streptozotocin induced diabetic rats. In the sub-chronic study OA and OA + insulin increased expression of GS in skeletal muscle of diabetic rats. GP expression was decreased by OA and OA + insulin treatments in skeletal muscle whilst in skin it was increased by both treatments. OA increased both GS and GP in skeletal muscle whilst in skin they were decreased. OA + insulin treatment increased GS and decreased GP activities in skeletal muscle and increased activity of both enzymes in skin of diabetic rats. OA increased the amount of glycogen in both muscle and skin whilst OA + insulin reduced the amount of glycogen. OA and OA + insulin treatment showed some protective effects against liver and muscle damage as there were reductions in serum LDH, ALT and AST levels. In conclusion, oleanolic acid in synergy with insulin can enhance activation of the insulin signalling pathway and there was evidence of OA activation of insulin signaling enzymes independent of insulin. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycaemia; this condition is caused by lack of insulin secretion (Type 1) and/or insulin resistance (Type 2). In diabetic patients; carbohydrate, protein and lipid metabolism is disturbed due to the lack of the body’s ability to utilise glucose efficiently. Management of type 1 diabetes involves insulin therapy which may be inconvenient for patients. Therefore alternative methods for management of type 1 diabetes involving medicinal products are being investigated. This study is aimed at investigating the effect of OA on markers of lipid metabolism and on proteins of the insulin signalling pathway in Type 1 diabetic rats as this plant product has anti-hyperglycaemic effects. Male Sprague-Dawley rats were divided into two groups (diabetic and normal). In both groups the rats were further divided into four groups and assigned to treatment as follows: vehicle, insulin, OA and OA plus insulin. Oral glucose tolerance test was performed in fasted and non-fasted diabetic rats for 2 hours. In acute studies the effect OA following treatment of rats was evaluated at 15, 30 and 60 minutes. In sub-chronic studies rats were treated daily for 14 days. OA did not improve glucose tolerance in diabetic rats after 2 hours of administration. However, it enhanced blood glucose lowering effect of insulin and this was statistically significant in fasted rats. In acute studies OA enhanced the effect of insulin in normal and diabetic animals as AKT phosphorylation was increased when insulin was used in combination with OA. OA reduced the expression and activity of HSL in liver tissue after 14 days of treatment in both normal and diabetic rats. In adipose tissue, OA reduced the expression of HSL in diabetic rats. However, OA alone did not reduce the activity of HSL but when it was combined with insulin, a reduction of HSL activity was observed. OA administration had no significant effect on TGA and HDL-c levels but significantly (p < 0.05) reduced total cholesterol and LDL-c in diabetic rats. It had no significant effect on total cholesterol, and increased LDL-c levels in normal rats. Serum AST and ALT levels in diabetic rats were reduced by OA administration but this reduction was not statistically significant. The results of this study suggest that OA enhances the hypoglycaemic effect of insulin, improves lipid profile and possesses hepatoprotective effects. Lastly, OA independently increases AKT phosphorylation and decreases HSL expression and activity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Thesis (Ph.D. (Dietetics))--North-West University, Potchefstroom Campus, 2010.

Thesis (Ph.D. (Dietetics))--North-West University, Potchefstroom Campus, 2010.

There are many tools available for the study of post-translational modifications. The majority of these tools is specific towards the individual modification and involves separation of modified proteins from non-modified ones. The drawback of using a modification specific method is that there is a lack of flexibility in its usage for other modifications. The goal of these studies was to investigate the possibility of obtaining a similar separation effect by fractionating post-translationally modified proteins based on the physical properties of proteins. The post-translational modification chosen to be the basis of this study was the O-GlcNAc modification. Using the C2C12 mouse myoblast cell line, it was determined that the optimal conditions for producing lysates containing increased yields of O-GlcNAc modified proteins was to treat differentiated C2C12 cells with 10nM insulin, 12g/L glucose and 2mM of the O-GlcNAcase inhibitor Streptozotocin for 24 hours. Using the optimized lysis buffer, it was shown that protein separation by surface charge using standard anion exchange separation did not provide enough resolution or material to obtain any identifications of modified proteins. However, when a chromatofocusing method which separates proteins on the basis of their isoelectric points was used, a separation scheme with larger capacity and higher resolution was possible. Using this separation method followed by gel electrophoresis of individual fractions, proteins which are potentially O-GlcNAc modified were identified by mass spectrometry. It was evident from the number of protein bands observed per fraction on the Coomassie stained gels and the number of proteins identified per protein band by mass spectrometry that further reduction in sample complexity was required to assist in the positive identification of O-GlcNAc modified proteins. Among the identified proteins, 32 percent were metabolic proteins, 21 percent were protein processing proteins, 16 percent were structural proteins and the remainder a mix of other proteins. Unfortunately, it was not possible to validate the presence or absence of the O-GlcNAc modification on these proteins using available methodologies such as immunoprecipitation. As such, further work is required to optimize the separation strategy and to verify the usefulness of this separation strategy in identifying O-GlcNAc/post-translationally modified proteins.

Orientador: José Roberto Sá Lima / Banca: Renato Paulo Chopard / Banca: Eduvaldo Silvino de Brito Marques / Resumo: O propósito deste trabalho foi estudar a reação do tecido ósseo após a implantação de polietileno poroso em defeitos cirúrgicos confeccionados no osso parietal de ratas diabéticas tratadas com calcitonina, mediante análise microscópica. Avaliou-se também a eficiência da aplicação da estreptozotocina na indução do diabete melito em ratos, em dose única de 45mg/Kg, através da utilização de um glicosímetro digital e do exame clínico. Foram utilizadas 27 ratas adultas, divididas em grupo não diabético-controle (C), grupo diabético (D) e grupo diabético tratado com calcitonina (DCa). Os animais do grupo DCa receberam aplicações subcutâneas do hormônio em doses de 16UI/Kg, em dias alternados, desde o pós-operatório imediato até o sacrifício e foram sacrificados após 15, trinta e sessenta dias. Diante dos resultados observados, pode-se concluir que o polietileno poroso foi tolerado pelos tecidos hospedeiros nos animais do grupo C e grupo DCa, provocando reações inflamatórias mais intensas nesse segundo grupo, em todos os períodos de observação. O polietileno poroso não foi tolerado pelos tecidos hospedeiros dos animais do grupo D. Os poros do implante foram preenchidos por um tecido conjuntivo bem vascularizado, conferindo estabilidade ao material no leito receptor, não ocorrendo osteointegração. A calcitonina utilizada nos animais do grupo DCa foi eficaz no tratamento pós-operatório dos animais diabéticos que receberam o polietileno poroso, inibindo o processo de reabsorção e/ou estimulando a neoformação óssea. A estreptozotocina, administrada em dose única de 45 mg/Kg, foi um método eficaz na indução do diabete melito nas ratas, permitindo boas condições de sobrevida. / Abstract: The purpose of this work was to study the reaction of the bone tissue after the porous polyethylene implantation in confectioned surgical defects in the parietal bone of diabetic rats treated with calcitonin, by means of microscopical analysis. The efficiency of a single dose of streptozotocin, 45mg/Kg, in the induction of diabetes mellitus in rats was also evaluated through the use of a digital glicosimeter and the clinical examination. Twenty-seven adult female rats had been used, divided in not diabetic-control (C), diabetic (D) and diabetic treated with calcitonin (DCa) groups. The animals of group DCa had received subcutaneous applications from the hormone in doses of 16U/Kg, in alternated days, since immediate postoperative until the sacrifice, and all the animals had been sacrificed after 15, 30 and 60 days. Ahead of the results, it can be concluded that the porous polyethylene was tolerated by the host tissue in groups C and DCa, disclosing more intense inflammatory response in group Dca. The pores of the implants had been filled with well vascularizated connective tissue, conferring stability to the material in the receiving site, not occurring osteointegration. Calcitonin used in the animals of group DCa was efficient in the postoperative treatment of the diabetic animals that had received the porous polyethylene implants, inhibiting the bone resorption and/or stimulating the bone formation. Streptozotocin, managed in a single dose of 45 mg/Kg, was an efficient method in the induction of the diabetes mellitus in the rats, allowing good conditions of survival. / Mestre

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University Of Technology 2014 / Oxidative stress (OS) plays a central role in the progression of diabetes mellitus (DM). Prevention of DM and its complications is a challenging health problem as it impacts on various organ functions, including reproduction. Diabetes mellitus with hyperglycaemic condition generates high production of reactive oxygen species. An imbalance between antioxidant mechanism and reactive oxygen species generates oxidative stress. OS damages the sperm membrane by oxidation of polyunsaturated fats which in turn reduces the sperm motility and ability to fuse with the oocyte and OS directly damage sperm DNA, compromising the paternal genomic contribution to the embryo development. Recent experimental evidence shows that modulation of oxidative stress and natural antioxidants may determine the outcome of male reproductive function. Previous investigations indicate that the supplementation and treatment with phytomedicine might play role in the prevention and management of DM and its subsequent complications on male reproductive function. This study explored the pharmacological potential of kolaviron (KV) on testicular and epididymal tissue in diabetic and non- diabetic Wistar rats. All experiments were conducted for a period of six weeks. Male Wistar rats (240–290 g) were randomly divided into 5 groups (n=12) where all the rats received a standard diet. Non diabetic rats control group and other four groups injected with different treatments. Non diabetic rat (N) received vehicle: Dimethylsulfoxide. Diabetes rats (D) were induced by a single intraperitoneal injection of freshly prepared streptozotocin (STZ) solution, 50mg/kg body weight. The N and D were treated with kolaviron (100 mg/kg body weight) orally, five times a week .The last group, diabetic rats were given subcutaneously injection of the standard anti-diabetic drug, insulin (0.2 u/kg) every second day. After the feeding period, testicular and epididymal tissues were collected and were analysed. All parameters were determined using appropriate methods in homogenized tissues. Data were expressed as mean ± SD. Plasma glucose as well as malondialdehyde (MDA) was significantly higher, while body, testicular and epididymal weights were lower in the D group compared to the N group and N+KV. Both kolaviron and insulin were able to ameliorate these effects. Testicular and epididymal antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in induced diabetic rats were significantly (p<0.05) low compared to diabetic control group. However, KV treated group shown significantly higher SOD, CAT and GPx activities compared D group. In conclusion, our findings demonstrated that KV could improve antioxidant enzymes and modulate STZ induced diabetic related oxidative stress in the male reproductive system. Kolaviron can potentially be used as an anti-diabetic treatment, however further studies are needed. Key words: Oxidative stress, Diabetes mellitus, antioxidants, kolaviron, epididymal tissue, testicular tissue, superoxide dismutase, catalase, glutathione peroxidase, lipid peroxidation, streptozotocin