The number of people having diabetes mellitus is increasing worldwide at an alarming rate. An unbalanced diet rich in carbohydrates and saturated fats, obesity and lack of physical activity, are being blamed. The worldwide prevalence of diabetes for all age-groups has been estimated to be 2.8% in 2000 and projected to be 4.4% by the year 2030. The pathogenesis of diabetes, especially the recent epidemic increase in type 2 diabetes, is still far from clear. Endothelial dysfunction, commonly defined as reduced endothelium-dependent relaxation due to reduced availability of the vasodilator mediator nitric oxide (NO), is a hallmark of diabetes mellitus. Methylglyoxal (MG) is a highly reactive dicarbonyl compound mainly formed as an intermediate during glycolysis. MG is also formed to a lesser extent from protein and amino acid metabolism. However, the relative contribution of various metabolic precursors to MG formation is not known. Levels of MG have been found to be elevated in diabetic and hypertensive conditions but it is not known whether MG is the cause or the effect of these pathological conditions. The aim of my project was (i) to quantify the amount of MG and oxidative stress produced from various substrates in cultured A10 vascular smooth muscle cells (VSMCs), (ii) to investigate the acute in vivo effects of a single dose of MG on glucose tolerance in male Sprague-Dawley (SD) rats, (iii) to investigate the effects of MG on endothelial function and (iv) to investigate the effects, and the underlying molecular mechanisms, of chronic administration of MG on glucose homeostasis in male SD rats. The results show that aminoacetone, a protein metabolism intermediate, is the most potent substrate for MG formation on a molar basis, whereas D-glucose and fructose are equipotent. I also established optimum sample preparation protocols for reproducible measurement of MG in biological samples by high performance liquid chromatography (HPLC). In normal SD rats a single acute dose of MG induced glucose intolerance, reduced adipose tissue glucose uptake and impaired the insulin signalling pathway, which was prevented by the MG scavenger and advanced glycation end product (AGE) breaking compound, alagebrium (ALT-711). MG and high glucose (25 mM) induced endothelial dysfunction in rat aortic rings and cultured endothelial cells by reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177, activity and NO production. MG and high glucose also increased oxidative stress and further reduced NO availability in rat aortic rings and cultured endothelial cells. Chronic administration of MG in normal SD rats by continuous infusion with a subcutaneously implanted minipump for 28 days (60 mg/kg/day), induced metabolic and biochemical abnormalities of glucose homeostasis and insulin regulation that are characteristic of type II diabetes. In MG treated rats, insulin stimulated glucose uptake in adipose tissue, and glucose stimulated insulin release from freshly isolated pancreas, were significantly reduced as compared to saline treated control rats. At a molecular level, insulin gene transcription was significantly impaired and apoptosis and DNA fragmentation were more prevalent in the pancreas of MG treated rats as compared to untreated control rats. All of these in vivo effects of MG were attenuated by the MG scavenger, alagebrium. Our data strongly indicate that MG is a causative factor in the pathogenesis of endothelial dysfunction and type 2 diabetes mellitus.

The number of people having diabetes mellitus is increasing worldwide at an alarming rate. An unbalanced diet rich in carbohydrates and saturated fats, obesity and lack of physical activity, are being blamed. The worldwide prevalence of diabetes for all age-groups has been estimated to be 2.8% in 2000 and projected to be 4.4% by the year 2030. The pathogenesis of diabetes, especially the recent epidemic increase in type 2 diabetes, is still far from clear. Endothelial dysfunction, commonly defined as reduced endothelium-dependent relaxation due to reduced availability of the vasodilator mediator nitric oxide (NO), is a hallmark of diabetes mellitus. Methylglyoxal (MG) is a highly reactive dicarbonyl compound mainly formed as an intermediate during glycolysis. MG is also formed to a lesser extent from protein and amino acid metabolism. However, the relative contribution of various metabolic precursors to MG formation is not known. Levels of MG have been found to be elevated in diabetic and hypertensive conditions but it is not known whether MG is the cause or the effect of these pathological conditions. The aim of my project was (i) to quantify the amount of MG and oxidative stress produced from various substrates in cultured A10 vascular smooth muscle cells (VSMCs), (ii) to investigate the acute in vivo effects of a single dose of MG on glucose tolerance in male Sprague-Dawley (SD) rats, (iii) to investigate the effects of MG on endothelial function and (iv) to investigate the effects, and the underlying molecular mechanisms, of chronic administration of MG on glucose homeostasis in male SD rats. The results show that aminoacetone, a protein metabolism intermediate, is the most potent substrate for MG formation on a molar basis, whereas D-glucose and fructose are equipotent. I also established optimum sample preparation protocols for reproducible measurement of MG in biological samples by high performance liquid chromatography (HPLC). In normal SD rats a single acute dose of MG induced glucose intolerance, reduced adipose tissue glucose uptake and impaired the insulin signalling pathway, which was prevented by the MG scavenger and advanced glycation end product (AGE) breaking compound, alagebrium (ALT-711). MG and high glucose (25 mM) induced endothelial dysfunction in rat aortic rings and cultured endothelial cells by reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177, activity and NO production. MG and high glucose also increased oxidative stress and further reduced NO availability in rat aortic rings and cultured endothelial cells. Chronic administration of MG in normal SD rats by continuous infusion with a subcutaneously implanted minipump for 28 days (60 mg/kg/day), induced metabolic and biochemical abnormalities of glucose homeostasis and insulin regulation that are characteristic of type II diabetes. In MG treated rats, insulin stimulated glucose uptake in adipose tissue, and glucose stimulated insulin release from freshly isolated pancreas, were significantly reduced as compared to saline treated control rats. At a molecular level, insulin gene transcription was significantly impaired and apoptosis and DNA fragmentation were more prevalent in the pancreas of MG treated rats as compared to untreated control rats. All of these in vivo effects of MG were attenuated by the MG scavenger, alagebrium. Our data strongly indicate that MG is a causative factor in the pathogenesis of endothelial dysfunction and type 2 diabetes mellitus.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

The introduction of high fructose corn syrup into the diet has been proposed to be the cause of many illnesses related to the metabolic syndrome. Fructose and its metabolites can be metabolized into cytotoxic reactive dicarbonyls that can cause damage to macromolecules leading to deleterious consequences. Dihydroxyacetone, a fructose metabolite, was studied in this thesis. Its ability to autoxidize and cause protein carbonylation under standard (pH 7.4, 37°C) and oxidative stress conditions (Fentons reagent) was investigated. Dihydroxyacetone was able to form significant amounts of dicarbonyls and protein carbonylation. Several selected natural polyphenols were chosen for an in vitro toxicological study involving rat hepatocytes. The chosen dietary polyphenols were rutin, gallic acid, methylgallate, ethylgallate, propylgallate and curcumin. In this thesis, the polyphenols were found to be able to significantly protect against the deleterious effects of glyoxal and methylglyoxal. In summary, these polyphenols could be candidates for future in vivo studies.

The introduction of high fructose corn syrup into the diet has been proposed to be the cause of many illnesses related to the metabolic syndrome. Fructose and its metabolites can be metabolized into cytotoxic reactive dicarbonyls that can cause damage to macromolecules leading to deleterious consequences. Dihydroxyacetone, a fructose metabolite, was studied in this thesis. Its ability to autoxidize and cause protein carbonylation under standard (pH 7.4, 37°C) and oxidative stress conditions (Fentons reagent) was investigated. Dihydroxyacetone was able to form significant amounts of dicarbonyls and protein carbonylation. Several selected natural polyphenols were chosen for an in vitro toxicological study involving rat hepatocytes. The chosen dietary polyphenols were rutin, gallic acid, methylgallate, ethylgallate, propylgallate and curcumin. In this thesis, the polyphenols were found to be able to significantly protect against the deleterious effects of glyoxal and methylglyoxal. In summary, these polyphenols could be candidates for future in vivo studies.

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound, formed as a metabolite from nonenzymatic and enzymatic reactions and is the leading precursor of advanced glycation end products (AGEs). AGEs contribute to ageing, type 2 diabetes mellitus (T2DM), and diabetes-related complications. However, MGO also has beneficial antibacterial activity and is the bioactive ingredient of medicinal honeys such as Manuka. Flavonoids are a group of phytochemicals that are powerful antioxidants. Polyphenols including flavonoids have been reported to trap MGO, forming adducts thereby preventing AGE formation. However, there is little to no information on the effect of adduct formation on the antioxidant properties of flavonoids and the antibacterial activity of MGO. In this study, catechin (CAT), chrysin (CHRY) and naringenin (NAR) at 0.1 mM and mixtures of each flavonoid with MGO (1:1) and (1:2) were evaluated for antioxidant and antibacterial activity. Antioxidant activity/capacity were evaluated with the total polyphenolic content (TPC), total flavonoid content (TFC), Trolox equivalent antioxidant capacity (TEAC) and the oxygen radical absorbent capacity (ORAC) assays. The bovine serum albumin (BSA)/MGO model was used to evaluate the effect on glycation. The 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) assay with the L929 cell line was used to evaluate cellular antioxidant activity. Cytotoxicity was determined in the L929 cell line using the crystal violet (CV) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assays. Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) were used to determine antibacterial activity using the microbroth-dilution assay and subsequent changes to morphology were evaluated using scanning electron microscopy (SEM). A reduction in antioxidant content was observed for: CHRY (TPC), CAT and NAR (TFC) and in antioxidant activity for: CHRY (TEAC) and CAT (ORAC), when combined with MGO. Overall most of the antioxidant activity of the flavonoids was not affected by the addition of MGO. In the presence of BSA and MGO, all flavonoid:MGO combinations reduced formation of AGEs except NAR in combination with MGO. All flavonoids alone and in combinations did not cause cellular oxidative damage while MGO and AAPH induced increased cellular damage indicating that MGO via AGE formation makes cells more sensitive to the effects of oxidants that form radicals. Only CAT reduced the oxidative effects of MGO/AAPH. For all combinations there was no effect on cell number, although cell viability was significantly reduced for CHRY and its combinations and for NAR and NAR:MGO1. Flavonoids at 0.1 mM CAT, CHRY and NAR had no antibacterial activity against E. coli while inhibition was observed only with NAR against B. subtilis. MGO at 0.1 and 0.2 mM inhibited bacterial growth while in combination the antibacterial activity was significantly reduced. MGO as well as NAR caused major changes to bacteria morphology. In combination, the antibacterial activity of MGO was reduced, and ultrastructure changes associated with toxicity was also observed in most groups. In conclusion, flavonoids do trap MGO and this effect does not significantly alter flavonoid antioxidant activity. However, the antibacterial activity of MGO is reduced. Future studies should focus on the chemistry and the effects involved and should include dosage dependent studies. / Dissertation (MSc)--University of Pretoria, 2019. / Anatomy / MSc / Unrestricted

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Ischemic cardiomyopathies, such as myocardial infarction (MI), are a leading cause of heart failure in both men and women throughout the world. Despite timely intervention post-MI, the loss of viable myocardium can lead to global remodeling and loss of function in many patients due to the limited regenerative potential of heart tissue. Thus, there is a critical need to better understand the repair mechanisms involved and to develop new preventative and reparative therapies for treating MI and preventing progression to heart failure. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite of glycolysis and the main precursor of advanced glycation end-products (AGEs), which can cause oxidative stress and wound healing delay. MG was shown to play an important causative role in the cellular changes, adverse remodeling and functional loss of the infarcted heart. This suggests MG as a target for therapy to restore cell-ECM signaling, inhibit oxidative stress and improve cardiac function post-MI. The aim of this PhD project was to develop new biomaterial therapies that can reduce the effects of MG, decrease oxidative stress, enhance electrical conductivity and improve cardiac contractility and function post-MI. There were three primary objectives: 1) To develop an injectable antioxidant and hydrogel system for minimizing the effects of MG and promoting cardiac repair post-MI; 2) To synthesize a nanoparticle system for targeted delivery of Glyoxalase-1 (Glo1) enzyme to cardiac tissue for reducing the accumulation of MG, limiting adverse remodeling and preserving cardiac function following MI; and 3) To design a sprayable nano-therapeutic that uses surface engineered custom designed multi-armed peptide grafted nanogold for on-the-spot coating of infarcted myocardial surface for increasing contractility of the myocardium post-MI. In the first study, a fisetin-loaded collagen type I hydrogel (fisetin-HG) was injected intramyocardially in mice at 3h post-MI, and compared to fisetin-alone, hydrogel-alone, or saline treatment. The fisetin-HG treatment increased the level of glyoxalase-1 (the main MG-metabolizing enzyme), reduced MG-AGE accumulation, and decreased oxidative stress in the MI heart, which was associated with smaller scar size and improved cardiac function. Treatment with fisetin-HG also promoted neovascularization and increased the number of pro-healing macrophages in the infarct area, while reducing the number of pro-inflammatory macrophages. The second study revealed that when delivered intravenously at 3h post-MI, our Glo1-loaded nanoparticles specifically targeted the damaged cardiac tissue, led to improved cardiac function, protected cell viability and limited infarct expansion by reducing oxidative stress post-MI. Lastly, the third study showed that, when applied at 1-week post-MI, the sprayed nanogold treatment remained at the treatment site for at least 28 days with no significant off-target organ infiltration. Our results demonstrated a remarkable increase in cardiac function, muscle contractility, and myocardial electrical conductivity post-MI. Overall, these findings show that reducing MG levels through both increased activity of Glo1 and direct MG scavenging as well as increasing cardiac contractility may be a promising approach to limit adverse cardiac remodeling, prevent damage, and preserve the function of the infarcted heart