Thesis submitted in fulfilment of the requirements for the Doctor of Technology: Biomedical Technology In the Faculty of Health and Wellness At the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY, 2012 / Diabetes mellitus is a major health problem not only in urban, but also in the rural areas and is diagnosed by the presence of high glucose levels in the blood. Oxidative stress is known to be actively involved in the onset and progression of diabetes and its complications. Antioxidants have important roles in biological systems by scavenging free radicals which may result in oxidative damage of biological molecules such as lipids, proteins and DNA. Red palm oil, originally from the tropical area of Africa, generally consumed as cooking oil, is known to have some beneficial health effects due to the presence of lipid soluble antioxidants such as carotenoids, tocopherols and tocotrienols. It also contains almost an equal proportion of both saturated and unsaturated fatty acids which makes it distinctive from other vegetable oils. Rooibos, on the other hand, is grown in the Cederberg area of the Western Cape in South Africa and it is commonly consumed as a beverage. It contains a complex profile of water soluble antioxidants (flavonoids) and its health promoting potentials have been reported extensively. Some of the flavonoids present in rooibos include aspalathin, nothofagin, quercetin, rutin and orientin. The objective of this research project was to examine the potential beneficial effects of the dietary intake of red palm oil and rooibos on streptozotocin-induced hyperglycaemia and its influence on the antioxidant status and some biochemical parameters in male Wistar rats. The preliminary phase of this study was designed to investigate the biochemical effects of these two plant products at different dosages following consumption for a period of 7 weeks. The preliminary study did not reveal any adverse effects of the different dosages of red palm oil (1 ml, 2 ml and 4 ml) and rooibos (2%, 4% and 6%) on the experimental rats following dietary intake for 7 weeks. However, these natural products showed an improvement in the antioxidant status of the rats at the different doses. Using a single dose each of both plant products from the preliminary study, the main study was performed to investigate the influence of these two plant products singly and in combination on the blood and liver of streptozotocin-induced hyperglycaemic male Wistar rats. In the main study, streptozotocin (50 mg/kg) dissolved in 0.1 M citrate buffer (pH 4.5) through intramuscular injection was used for the induction of diabetes which was confirmed by the presence of high blood glucose after 72 hours. Red palm oil or rooibos extract alone did not have any effect on the control of blood glucose in the diabetic rats. The dietary intake of the combined treatment with red palm and rooibos had more health promoting effects on the diabetic rats which included a decrease in blood glucose, glycosylated haemoglobin, fructosamine and increased insulin levels. There was a marked increase in liver glycogen levels in all the diabetic groups. Treatment with rooibos alone showed a decrease in glycogen levels in the diabetic rats. The presence of liver enzymes in the serum, commonly used as indicators of liver damage was increased in all the diabetic rats. However, the combined treatment of diabetic rats with red palm oil and rooibos protected the liver from injury. Red palm oil improved high density lipoprotein cholesterol levels (HDL-cholesterol) in the diabetic rats. There was no effect on the activity of glucokinase, the first enzyme in the the glycolytic pathway in both the untreated and treated diabetic rats. However, the activity of pyruvate kinase, the last enzyme in the glycolytic pathway was reduced in all the diabetic groups. The combined treatment with both red palm and rooibos increased the activity of pyruvate kinase. Oxidative stress was confirmed in the diabetic rats with an increase in the plasma thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation. Treatment of diabetic rats with rooibos and the combination of red palm oil and rooibos brought plasma TBARS to a level that was not significantly different from the normal control group. There was a non-significant reduction of total glutathione in the non-treated and treated diabetic groups. A non-significant increase in the activity of liver catalase was observed in all the treated diabetic groups. The activity of superoxide dismutase was significantly decreased in the liver of diabetic rats. Diabetic rats treated with red palm oil, rooibos and the combined treatment showed an increased activity of superoxide dismutase in the liver. Red palm oil and the combined treatment increased the activity of glutathione peroxidase in both the red blood cells and liver of diabetic rats. Red palm oil, rooibos and their combined treatments also improved the plasma antioxidant capacity such as ferric reducing antioxidant power (FRAP) and oxygen reducing absorbance capacity (ORAC) in the diabetic rats. In conclusion, oxidative stress is actively involved in the progression of diabetes mellitus. Red palm oil and rooibos, most especially their combined treatment showed significant beneficial health promoting effects in the diabetic rats. The remarkable effects of the combined treatment of red palm oil and rooibos in the diabetic rats could be due to their antioxidant profiles. Based on the findings from this study, it can be adduced that these plant products could help in the management of diabetes and its complications and therefore, suggested the need for further research studies on antioxidant therapy in the management of diabetes mellitus.

Thesis (DTech (Biomedical Technology))--Cape Peninsula University of Technology, 2017. / Diabetes mellitus (DM) is characterized by deficiency in insulin resulting in hyperglycaemia with metabolic alterations in carbohydrate, lipid and protein. DM has been associated with increased formation of reactive oxygen species (ROS) and inflammatory mediators. Many drugs have been designed for its treatment and management; however, limitations persist in the use of anti-hyperglycemic medications due to numerous side effects, high cost, limited action and secondary failure rates. Moringa oleifera (MO) tree is distributed in the tropics and subtropics and has been found to be very nutritious with a variety of applications. This plant has been reported to possess antidiabetic, antioxidant and other medicinal properties which may be helpful in managing diabetes and its associated complications. This study investigated the antioxidant status, antidiabetic, antilipidemic, anti-inflammatory, anti-apoptotic properties and phytochemical constituents of the leaf extract of MO (250 mg/kg). Diabetes was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (STZ) in buffered citrate (0.1, pH 4.5). Forty-eight Wistar rats were randomly divided into four (4) groups and treated for six weeks: group one- non-diabetic control (Control), group two- non-diabetic Moringa treated (Moringa), group three- diabetic control (Diabetic) and group four- diabetic Moringa treated (Diabetic + Moringa). Methanol, aqueous and petroleum ether extract of MO leaves were evaluated for its antioxidant and phytochemical contents. Assays for total antioxidant capacity such as trolox equivalence antioxidant capacity (TEAC), oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), flavonoids, flavonols and total polyphenols content were analysed. Other parameters analysed include glucose level; glycated haemoglobin level; hepatic biomarkers; endogenous antioxidants (SOD, CAT, GSH, GPx) in the liver; kidney and erythrocytes; inflammatory biomarkers in the serum, liver and kidney; high-density lipoprotein (HDL), low-density lipoprotein (LDL) and total cholesterol (TC) in serum. Assessment of apoptotic cell death biomarkers (caspase 3, caspase 9, BCL-2, NFKβ, p53) in the liver and kidney were performed. Histopathological analysis was conducted on the liver, kidney and pancreatic sections. In vitro results showed that aqueous and methanol extract of MO demonstrated a high antioxidant capacity, phenolic contents and revealed more chemical constituents than the petroleum ether extract. HPLC analysis of the leaf extract indicated the presence of flavonoids: quercetin, rutin and myricetin and phenolic acids. High levels of polyphenols, flavonols and alkaloids were reported in MO extracts. Treatment with MO in normal and diabetic rats daily for six weeks resulted in significant (p<0.05) decrease in glucose and glycated haemoglobin levels. Liver and kidney size which increased in diabetic rats, decreased significantly (p<0.05) after treatment with MO. Pancreas size showed significant (p<0.05) decrease in diabetic rats and increased significantly (p<0.05) after MO administration. Similarly, serum albumin level increased in non-diabetic and diabetic groups after MO treatment. Also, a significantly increased level of T-bilirubin in diabetic groups relative to normal control rats which reduced greatly after MO administration was observed. Serum lipid profile: LDL and TC levels were increased in rats exposed to STZ. HDL level decreased in diabetic rats when compared to normal control. The activities of MO extracts was shown to lower TC and LDL levels. HDL level also increased after MO administration. Similarly, lipid peroxidation (MDA) level significantly (p<0.05) decreased in the diabetic group following MO treatment. An observable improvement was seen in the antioxidant enzyme system. Activities of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and concentration of glutathione (GSH) were restored or increased in the homogenate of the liver, kidney, and erythrocytes, indicative of the protective effect of MO in diabetic and non-diabetic rats. The expression of cell death markers (caspase 3, caspase 9, BCL-2, NFKβ, p53) showed remarkable improvement after treatment with MO relative to the non-diabetic control. A significant (p<0.05) reduction in inflammatory cytokines (IL-1α, IL-6, IL-12, IL-18, TNF-α) and (chemokine MCP-1 concentrations) were observed in the serum, liver, and kidney of non-diabetic and diabetic treated groups. Histopathological sections of the liver, kidney and pancreas of diabetic rats revealed severe damage which showed significant improvements after MO treatment. Liver, kidney and pancreatic histological sections revealed the protective effect of MO in both non-diabetic and diabetic rats. MO exerted modulatory effects in STZ-induced diabetes by its antidiabetic, hypoglycemic, antioxidant, anti-inflammatory, anti-apoptotic and anti-lipidemic activities and offered protective effects against diabetic-induced nephrotoxicity and hepatotoxicity, but equally improved antioxidant status. The study concluded that MO could play a significant role in the early treatment and management of diabetes that pharmaceutical industry should consider it in the future as a possible therapeutic agent.

Orientadora: Profª. Drª. Tatiana Lima Ferreira / Coorientador: Prof. Dr. Daniel Carneiro Carrettiero / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, São Bernardo do Campo, 2018. / A doença de Alzheimer (DA) é uma doença neurodegenerativa caracterizada por múltiplos déficits cognitivos, como prejuízo da memória operacional, espacial e emocional. As principais características histopatológicas da DA são o surgimento das placas amilóides e dos emaranhados neurofibrilares. Os emaranhados se originam do acúmulo da proteína tau fosforilada (p-tau), que ocorre devido a falhas nas suas vias de degradação, como a via BAG2/HSP70. Esse acúmulo de p-tau no progresso da DA é parte fundamental do surgimento e agravamento de processos neurodegenerativos como perda sináptica e neuronal, relacionada à piora do comprometimento cognitivo. Os compostos canabinóides tem importante papel no progresso da DA, regulando processos cognitivos, inflamatórios e de degradação de p-tau. Uma redução da quantidade de anandamida (ANA) um endocanabinóide, no encéfalo de humanos está correlacionada com o déficit cognitivo associado à DA. Além disso, uma das formas da ANA exercer seu papel neuroprotetor em processos neuropatológicos seria aumentando os níveis de HSP70, chaperona envolvida na degradação de p-tau. A maioria dos estudos que investigo Os animais que receberam STZ tiveram déficits nos testes de reconhecimento de novo objeto (RNO), latência de escape no labirinto em cruz elevada (LCE) e condicionamento de medo ao som e a injeção de ANA recuperou os prejuízos no RNO e no LCE. O condicionamento de medo ao contexto e a inibição pré-pulso não foram alterados pela STZ e/ou ANA. STZ também induziu alargamento ventricular, redução de receptores canabinóides do tipo 1 e das proteínas sinápticas sinaptofisina e sintaxina. ANA preveniu o aumento do ventrículo e a diminuição de sintaxina. Os níveis de SNAP-25 não foram modificados por ANA e/ou STZ. A respeito da via de degradação da ptau, STZ aumentou os níveis de p-tau Ser 396 e diminuiu p-tau Ser199/202, HSP70 e BAG2. A ANA não preveniu nenhum desses efeitos, apenas acentuou a redução de BAG2. A quantidade de tau total permaneceu inalterada pelos tratamentos. Os resultados sugerem que o sistema endocanabinóide pode estar envolvido nos efeitos induzidos pela STZ, pois, embora a ANA não tenha conseguido prevenir algumas alterações cognitivas observadas na DA esporádica, ela evitou parcialmente as deficiências cognitivas induzidas pela STZ e as alterações no volume ventricular e nos marcadores sinápticos. A relação desse sistema com a degradação de p-tau ainda permanece não esclarecida e são necessários mais estudos para melhor compreensão do substrato neurobiológico pelo qual a ANA poderia exercer sua atividade neuroprotetora. .Assim, este estudo fortalece o uso de STZ como modelo de DA esporádica e corrobora o potencial do sistema endocanabinóide como um importante alvo terapêutico para a DA. / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by multiple cognitive deficits, such as impairment of working, spatial and emotional memory. The main histopathological features of AD are the appearance of amyloid plaques and neurofibrillary tangles. The tangles are originated from the accumulation of phosphorylated tau protein (p-tau), which occurs due to failures in its degradation mechanisms, such as the BAG2/HSP70 pathway. This accumulation of p-tau in AD progress is a fundamental part of the onset and aggravation of neurodegenerative processes, including synaptic and neuronal loss, related to the worsening of cognitive impairment. Cannabinoid compounds play an important role in the pathological progression of AD, regulating cognitive and inflammatory processes and p-tau degradation. A reduction in the amount of anandamide (ANA), an endocannabinoid, in the brain of humans is correlated to the cognitive deficit associated with AD. In addition, one of the ways ANA exert its neuroprotective role in neuropathological processes would be by increasing the levels of HSP70, chaperone involved in the degradation of p-tau. Most studies investigating the role of the endocannabinoid system in AD have used transgenic animals, which are more correlated to familial AD, and are not ideal models to mimic sporadic AD, which is the multifactorial form of the disease, corresponding to more than 95% of the cases. The sporadic DA model based on the intracerebroventricular (icv) injection of streptozotocin (STZ) mimics the cognitive, metabolic, synaptic and histopathological changes of the AD. However, little is known about the role the endocannabinoid system in the changes observed in this model. Thus, the objective of the present study was to evaluate the possible preventive effect of ANA on cognitive changes, synaptic loss and alterations in the HSP70/BAG2 pathway of p-tau degradation in a sporadic AD model. Thus, Wistar rats received ANA and/or STZ icv in the same surgery and after 30 days had their cognitive performance evaluated. Afterwards, they were euthanized and their brains were removed for histological analysis and the hippocampus were used for Western blotting analysis of the onditioning tests and ANA injection recovered the impairments of the performance in NOR and EPM. Contextual fear conditioning and pre-pulse inhibition were not altered by STZ and/or ANA. STZ also induced ventricular enlargement and reduced the levels of cannabinoid receptors of type 1 and of the synaptic proteins synaptophysin and syntaxin. ANA prevented ventricular enlargement and the decrease of syntaxin levels. SNAP-25 levels were not modified by ANA and/or STZ. Regarding the degradation pathway of p-tau, STZ increased p-tau Ser 396 and decreased p-tau Ser199 / 202, HSP70 and BAG2. ANA did not prevent any of these effects, only accentuated the reduction of BAG2. The total levels of tau remained unchanged by the treatments. The results suggest that the endocannabinoid system may be involved in the STZ-induced effects because, although ANA failed to prevent some cognitive changes in sporadic AD, it partially inhibited STZ-induced cognitive deficits and changes in ventricular volume and in the synaptic markers. The relationship of this system to the degradation of p-tau remains unclear and further studies are needed to better understand the neurobiological substrate by which ANA could exert its neuroprotective activity. Thus, this study strengthens the use of STZ as a sporadic DA model and corroborates the potential of the endocannabinoid system as an important therapeutic target for AD.

>Magister Scientiae - MSc / About 180 million people have been estimated to suffer from type 2 diabetes (T2DM) in 2006 and the annual death rate due to this disease was 3 million by that time. More than 400 medicinal plants used for the treatment of diabetes mellitus have been recorded, but only a small number of these plants have received scientific and medical evaluation to assess their efficacy. The most common plant used to treat diabetes mellitus is Hypoxis hemerocallidea (HH). The present study was undertaken to investigate the effects of Hypoxis hemerocallidea (HH) on T2DM in rats. Male Wistar rats weighing 200-250 g were used in this experiment. Hypoxis hemerocallidea (HH) corm was used as plant material in the experiment. The study was based on three parts, an acute diabetes study, chronic diabetes study and insulin secretion study. In the acute study, the rats were randomly divided into 2 groups (control and diabetes). The saline solution was added to different concentrations of HH corm to produce concentration of (50, 200, 400, 800 mg/ml). Diabetes was induced by intraperitoneal injections of STZ (65mg/kg). Two weeks after the injection (STZ 65 mg/kg), different concentrations of HHS was administered intraperitoneally after an overnight fast. The blood glucose levels were monitored in the diabetic and control rats at, 30, 60, 120, 180 and 240 minutes post injection. In the chronic study, the rats were randomly divided into 6 different groups (control, HFD, DM, DM-HH, DM-PTHH, and HH). Diabetes mellitus was then induced in the groups of diabetic rats by intraperitoneal injections of STZ (40 mg/kg) and rats were fed a high fat diet (HFD). The body weight of the rats were measured weekly for 7 weeks. An intraperitoneal glucose tolerance test (IPGTT) was performed at the end of week 6. At the end of week 7, rats were killed and serum sample were collected for determination of fatty acid and insulin. Liver and pancreatic tissue was collected for histological evaluation. In the insulin secretion study, Hypoxis hemerocallidea was tested for its effects on insulin secretion by pancreatic islet cells exposed to low (3mM) and high (20mM) glucose medium. Results of the acute study indicated that HHS at a dose 800 mg/ml decreased blood glucose levels fastest in both normal and diabetic rats reaching significance after 30 minutes and 60 minutes respectively and remained below the baseline value until 240 minutes. In the chronic study, it was illustrated that HH had no effect in normal rats on any of the parameters evaluated. Animals in the DM group gained weight the first two weeks, but thereafter began to lose weight. At the end of seven weeks the animals gained significantly less weight than the rest. Animals fed a HFD have more visceral fat compared to the control group. The visceral fat gain occurred in the absence of a significant increase in body weight. We found a markedly lower fasting glucose level in HH treated diabetic animals compared to untreated DM animals. At time zero the blood glucose level of the HFD group (5.8±0.5mmol/l) and the HH group (4.9±0.7mmol/l) were in the normal range, and were not significantly different (P > 0.05) from the control group (5.0±0.2mmol/l). After glucose load peak blood glucose levels was measured after 30 minutes in the control group (9.0±0.6mmol/l), the HFD group (9.8±0.4 mmol/l), the DM-HH group (21±5.7 mmol/l) and the DM-HHPT group (27.8±5.3 mmol/l). In the HH group the blood glucose level reached a peak at 60 minutes (7.6±0.6 mmol/l). In the DM group two peaks were recorded one after 10 minutes (27.2±7.1mmol/l) and another after 60 minutes (31±5.2 mmol/l). In the groups control, HFD, DMHH, DM-HHPT and HH groups the blood glucose level after 120 minutes were not significantly different from the time zero value. The blood glucose level after 120 minutes in the DM group (28.2±7.1 mmol/L) was significantly higher (P ≤ 0.01) than from the time zero value. Serum fatty acid levels were increased in all groups fed a high fat diet. The serum insulin levels in the HFD group (6.2 ± 0.76 μUI/ml protein; P ≤ 0.05 ), the DM group (2.0 ± 0.9 μUI/ml protein; P ≤ 0.001), the DMHH group (3.4 ± 0.7 μUI/ml protein; P ≤ 0.001) and the DM-HHPT group (3.0 ± 1.1 μUI/ml protein; P ≤ 0.001) were significantly lower than the control group. The β-cell function in the HFD group (62 ± 8 %; P ≤ 0.001), the DM group (3 ± 1 %; P ≤ 0.001), the DM-HH (11 ± 9 %; P ≤ 0.001) group and the DM-HHPT group (4 ± 2 %; P ≤ 0.001) were significantly lower than the control group. The histological observation of the liver and the pancreas in rats after 7 weeks on different dietary regimes showed some morphological changes within the liver and pancreas parenchyma of some rats. In the insulin secretion study, glucose stimulated insulin secretion in low (3mM) and high (2mM) glucose concentration. Furthermore, insulin secretion was significantly higher when the glucose concentration was increased from 3mM to 20 mM (1.10 ± 0.13 μUI/ml protein and 1.5 ± 0.17 mIU/mg protein respectively P≤ 0.01). In the presence of low HH (100 µg/ml), there was a marked increase in insulin secretion when exposure to high glucose compared to low glucose concentration, while in the presence of high HH (500 µg/ml), there was no significant different in insulin secretion in the presence of low or high glucose. In conclusion, the results of this experimental study indicate that a concentration 800 mg/kg of HHS produces maximal hypoglycaemic effect in fasted normal and diabetic rats. HH has an antidiabetic activity as it lowers serum glucose levels in T2DM rats and significantly increases glucose tolerance. It also increases body weight of diabetic rats. HH treatment was found to improve insulin secretion in pancreatic islet cells.

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. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

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.

Orientadores: Marcelo Alexandre Prado, Débora Barbosa Vendramini Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-27T19:08:56Z (GMT). No. of bitstreams: 1 Oliveira_MicheleChristineMachadode_M.pdf: 3902978 bytes, checksum: 4d585a98c3af2e67dbde235b37799309 (MD5) Previous issue date: 2015 / Resumo: Estudos envolvendo compostos antioxidantes presentes em alimentos e a prevenção ou controle de algumas doenças não transmissíveis têm chamado a atenção da comunidade científica e da população em geral, considerando que esses estudos abrem novas possibilidades para a descoberta de novas substâncias bioativas. Entre os alimentos que contém antioxidantes naturais, as sementes constituem uma importante fonte de suprimento dietético. Dentre as sementes utilizadas pela população para fins medicinais está o alpiste (Phalaris canariensis L.), tradicionalmente usado como tratamento alternativo para o diabetes, porém são escassos os estudos científicos conduzidos com essa espécie. Dessa forma, o objetivo desse estudo foi avaliar a composição química e a atividade antioxidante e hipoglicemiante do extrato aquoso de alpiste. A composição das sementes e do extrato aquoso foram realizados segundo as normas e metodologias da AOAC e Instituto Adolfo Lutz. Os métodos empregados para a avaliação da atividade antioxidante foram o ABTS (2,2'-azino-bis-3-etilbenzotriasolina- 6-ácido sulfônico), DPPH radical (2,2-difenil-1-picrilhidrazil) e o ORAC (Oxygen Radical Absorbance Capacity). A avaliação da atividade hipoglicemiante foi realizada por meio do modelo de diabetes induzida por estreptozotocina (STZ, dose única de 60 mg/kg, via intraperitoneal) em ratos Wistar machos, que foram aleatoriamente distribuídos em grupos de 10 animais, sendo: sham (animais sadios não-diabético); controle negativo (diabéticos não tratados), tratados com extrato aquoso de alpiste (doses de 250, 500 e 1000 mg/kg, via oral) e não diabéticos tratados com a dose de 1000 mg/kg. Dois protocolos foram realizados: tratamentos por 28 dias e de longa duração por 87 dias. Em ambos protocolos os animais foram monitorados semanalmente quanto a massa corporal, glicemia e consumo de água e ração. No final dos experimentos os órgãos foram removidos e pesados e sangue e urina foram coletados para avaliações bioquímicas, hematológicas, eletrolíticas e histopatológicas, afim de constatar a ação do extrato aquoso de alpiste no diabetes. Os resultados obtidos nas sementes de dois lotes e do extrato aquoso foram respectivamente: umidade e resíduo seco (10,31%; 9,50%; 78,21%), cinzas (6%; 5,30%; 1,74%), proteínas (14,88%; 15,12%; 18,26%), lipídeos (5,38%; 5,17%; 2,07%), amido (50,54g/100g; 48,04g/100g; 3,79g/100g) e fibras totais (18,88g/100g; 17,29g/100g; 0,70g/100g). Os ácidos graxos encontrados predominantes foram: ácido palmítico (12%) e poli-insaturados: linoleico (53%), oleico (28%) e linolênico (3%). Compostos fenólicos totais (280,15 ± 3,05 µg EAG/g) e Atividade Antioxidante: ABTS (228,93 ± 2,25µg eqtrolox/g), DPPH (106,17 ± 6,69 µg eqtrolox/g) e ORAC (1177,37 ± 5,32 µM/g na fração hidro e 147,79 ± 0,48 µM/g na lipo). O alpiste e seu extrato mostraram potencial nutritivo, e o extrato apresentou atividade antioxidante intermediária. O tratamento com extrato aquoso de alpiste nas doses experimentais não controlou os níveis glicêmicos, bem como não apresentou efeitos sobre a massa corporal, consumo de água e ração e em nenhum dos parâmetros bioquímicos e hematológicos avaliados, evidenciando que este extrato não apresenta efeitos antiabetogênicos / Abstract: Studies concerning the application of antioxidant compounds from food in the prevention or control of non-transmissible diseases attracted attention of the scientific community and population in general, as theses studies open new possibilities for the discovery of new bioactive compounds. Among foods that contain natural antioxidants, the seeds are an important source of dietary supply. Among the seeds used by the population for medicinal purposes is the canary seed (Phalaris canariensis L.), traditionally used as an alternative treatment of diabetes, however there are only few studies concerning the biological actions of this specie. Thus, the aim of this study was to evaluate the chemical composition and the antioxidant and hypoglycemic activities of the aqueous extract from canary seed. The chemical composition of the extract and seeds was performed according to the methodology and standards of AOAC and Adolfo Lutz Institute. The used to evaluate the antioxidant activity were ABTS (2,2'-azino-bis-3-etilbenzotriasolina- 6-ácido sulfônico), DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (Oxygen Radical Absorbance Capacity). For the evaluation of the hypoglycemic activity, the streptozotocin-induced diabetes model was conducted (STZ, single dose of 60 mg/kg, intraperitoneal route) in male Wistar rats, which were randomly divided into groups of 10 animals, such as: sham (healthy animals, non-diabetic), negative control (diabetic, untreated), treated with aqueous extract from canary seeds (250, 500 and 1000 mg/kg, oral route, daily) and non-diabetic treated with 1000 mg/kg of the extract. Two protocols were performed: treatments for 28 days and for 87 days. In both experiments, the animals were weekly monitored for body weight, glycemia and consumption of food and water. In the end of the experiments, organs were removed and weighted and blood and urine were collected for biochemical, electrolytic and histopathological evaluations, in order to evaluate the action of the aqueous extract of canary seeds in diabetes. Results obtained for seeds obtained from two different lots and for the extract were: humidity and dry residue (10.31%; 9.50%; 78.21%), ash (6%; 5.30%; 1.74%), proteins content (14.88%; 15.12%; 18.26%), lipid content (5.38%; 5.17%; 2.07%), starch content (50.54g/100g; 48.04g/100g; 3.79g/100g) and total fibers (18.88g/100g; 17.29g/100g; 0.70g/100g). Fatty acids were predominantly: palmitic acid (12%) and polyunsaturated: linoleic (53%), oleic (28%), linolenic (3%). Total phenolic compounds (280.15 ± 3.05 µg EAG/g) and antioxidant activity: ABTS (228.93 ± 2.25µg eqtrolox/g), DPPH (106.17 ± 6.69 µg eqtrolox/g) and ORAC (1177.37 ± 5.32 µM/g in the hydro fraction and 147.79 ± 0.48 µM/g in the lipidic fraction). In sum, the aqueous extract from canary seeds showed a nutritional potential and presented intermediate antioxidant activity. Treatments with the extract in the experimental doses did not control the glycemic levels, as wells as had no effects in the body weight, consumption of food and water and in any of the biochemical and hematological evaluations, thus evidencing that the aqueous extract from canary seeds does not have antidiabetogenic effects / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

Objective: Atherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s). Methods and Results: Wild-typemice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less a-SMA-positive area) and had more inflammation (increased plasma IL-1b level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemicmice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks. Conclusion: We established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice.

Background: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a propancreatic endodermal transcription factor, iPS cells, or their respective conditioned media (CM) on diabetic cardiomyopathy. Methods: Experimental diabetes was induced in male Sprague Dawley rats (8-10 weeks old) by intraperitoneal STZ injections (65 mg/kg body weight for 2 consecutive days). Animals were divided into six experimental groups including control, treated with sodium citrate buffer IP, STZ, STZ + ES-Ptf1a cells, STZ + iPS cells, STZ + ES-Ptf1a CM and STZ + iPS CM. Following STZ injections, appropriate cells (1 X 106/mL/injection/day) or CM (2 mL injection/day) were given intravenously for 3 consecutive days. Animals were sacrificed and hearts were harvested at day 28. Histology, TUNEL staining, and Caspase-3 activity were used to assess apoptosis and fibrosis. ERK1/2 phosphorylation was quantified using ELISAs. M-mode echocardiography fractional shortening was used to assess cardiac function. Results: Animals transplanted with ES cells, iPS cells, or both CMs showed a significant (p

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