Flavanols are polyphenols that are known to have many beneficial effects on the body. Cocoa is a major source of these flavanols. However, research on the potential anti-obesity and anti-diabetic health benefits of cocoa flavanols is lacking in the literature. Furthermore, the effect that the size of these flavanols has on the extent of its beneficial properties is also unclear. The objective of this study was to evaluate the dietary effects of cocoa flavanols on the onset of obesity, insulin resistance and impaired glucose tolerance and to determine the impact that the size of these compounds has on the magnitude of this effect. Cocoa extract was fractionated into a monomer-, an oligomer-, and a polymer-rich fraction. Six groups (n=9) of C57BL/6J mice were fed either a control low-fat diet, a control high-fat diet, or a high-fat diet supplemented with 25 mg/kg*BW of cocoa extract or one of the three cocoa fractions. After 12 weeks on these diets, the oligomer-rich fraction proved to be most effective in preventing weight gain, fat mass accumulation, elevated fasting blood glucose and impaired glucose tolerance in diet-induced obese mice. This is the first long-term feeding study to examine the relative activities of cocoa constituents on diet-induced obesity and insulin resistance. / Master of Science in Life Sciences

Yes / Insulin resistance is associated with impaired endothelial regeneration in response to mechanical injury. We recently demonstrated that insulinlike growth factor–binding protein-1 (IGFBP1) ameliorated insulin resistance and increased nitric oxide generation in the endothelium. In this study, we hypothesized that IGFBP1 would improve endothelial regeneration and restore endothelial reparative functions in the setting of insulin resistance. In male mice heterozygous for deletion of insulin receptors, endothelial regeneration after femoral artery wire injury was enhanced by transgenic expression of human IGFBP1 (hIGFBP1). This was not explained by altered abundance of circulating myeloid angiogenic cells. Incubation of human endothelial cells with hIGFBP1 increased integrin expression and enhanced their ability to adhere to and repopulate denuded human saphenous vein ex vivo. In vitro, induction of insulin resistance by tumor necrosis factor α (TNFα) significantly inhibited endothelial cell migration and proliferation. Coincubation with hIGFBP1 restored endothelial migratory and proliferative capacity. At the molecular level, hIGFBP1 induced phosphorylation of focal adhesion kinase, activated RhoA and modulated TNFα-induced actin fiber anisotropy. Collectively, the effects of hIGFBP1 on endothelial cell responses and acceleration of endothelial regeneration in mice indicate that manipulating IGFBP1 could be exploited as a putative strategy to improve endothelial repair in the setting of insulin resistance. / Funded by a British Heart Foundation Clinical Research Training Fellowship for A.A. R.M.C. holds a British Heart Foundation Intermediate Clinical Research Fellowship. M.T.K. holds a British Heart Foundation Chair in Cardiology. S.B.W. holds a European Research Council Starting Grant.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: A westernized diet rich in saturated fats and sugars, together with a sedentary lifestyle, has contributed to the dramatic increase in obesity during the last decade (Zimmett et al, 2001; Wild et al, 2004). Obesity is associated with dyslipidemia and insulin resistance which are major risk factors for the development of type 2 diabetes (T2D) (Zimmet et al, 2001, Kahn et al, 2006; Schröder et al, 2007). High-fat feeding in rodents induces symptoms similar to the human metabolic syndrome without progression to T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). The addition of fructose to a high-fat diet exacerbates the insulin resistance and leads to impaired pancreatic function of insulin secretion and glucose intolerance (Basciano et al, 2005; Stanhope et al, 2009). Aims: The aim of this study was to establish the effect of a high-fat and sucrose/fructose diet on glucose metabolism, the development of insulin resistance and β-cell dynamics. Methods: Weanling Wistar rats were randomized into two study groups; study one over an experimental period for three months and study two for twelve months. Each study consisted of a control group that received standard rat chow and water; and two experimental groups receiving either a high-fat diet and water (HFD) or a café diet consisting of HFD with the addition of 15% sucrose/fructose (CFD). Fasting glucose and insulin concentrations, intravenous glucose tolerance test (IVGTT), glucose stimulated insulin secretion rates and 2-deoxy-[3H]-D-glucose uptake in muscle, liver and fat were measured. The pancreata were harvested for immunohistochemical labeling of β-cells (insulin), α-cells (glucagon), GLUT2 (glucose transport) and MIB5 (proliferation). Samples of the pancreata were also collected for electron microscopy. Results and discussion: Feeding Wistar rats a CFD induced obesity, insulin resistance and glucose intolerance. By twelve months the rats had an impaired glucose response with increased IVGTT peak values, area under the curve (AUC) values and glucose clearance rates. Concomitantly, the glucose stimulated insulin secretion rate (GS-ISR) was attenuated. Stimulated glucose disposal as measured by 2-deoxy-[3H]-D-glucose uptake was reduced in muscle and adipose tissue at three months. By twelve months, due to the age of the rats, stimulated glucose uptake declined compared to three months with no difference between groups. After three months the diets had no observable effect on the islets using light microscopy. However, by twelve months morphological changes were observed in both the HFD and CFD groups. In the HFD group large hypertrophied irregular islets with fibrous changes were observed. In the CFD group these morphological changes were more prominent with fibrous segregation and disruption of the normal endocrine arrangement. In addition, the presence of inflammatory cells within the affected islets is consistent with T2D. Conclusion: High-fat diet fed to Wistar rats induced obesity, abdominal adiposity and insulin resistance. The addition of sucrose/fructose to a high-fat diet exacerbated the insulin resistance and resulted in glucose intolerance and mild hyperglycemia. Morphological changes in the large islets were observed which are consistent with the development of T2D. / AFRIKAANSE OPSOMMING: Inleiding: ‘n Verwesterde dieët, ryk aan versadigde vette en suikers tesame met 'n passiewe lewenstyl, het bygedra tot die dramatiese verhoging in vetsug gedurende die laaste dekade (Zimmett et al, 2001; Wild et al, 2004). Vetsug word met dislipidemie en insulienweerstandigheid geassosieer wat hoof risikofaktore is vir die ontwikkeling van tipe 2 diabetes (T2D) (Zimmet et al, 2001; Kahn et al, 2006; Schröder et al, 2007). Hoë-vet voeding in knaagdiere induseer simptome soortgelyk aan menslike metaboliese sindroom sonder die ontwikkeling van T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). Die byvoeging van fruktose tot 'n hoë-vet dieët vererger insulienweerstandigheid en lei tot verswakte pankreas funksie, insuliensekresie en glukoseintoleransie (Basciano et al, 2005; Stanhope et al, 2009). Doelwitte: Die doelwitte van die studie was om die effek van hoë-vet en sukrose/fruktose voeding op glukosemetabolisme, die ontwikkeling van insulienweerstandigheid en β-sel dinamika te bepaal. Metodes: Gespeende Wistar rotte was in twee groepe gerandomiseer; studie een oor ʼn tydperk van drie maande en studie twee oor ʼn tydperk van twaalf maande onderskeidelik. Elke studie het 'n kontrole groep met standaard rot kos en water (control); en twee experimentele diëte wat of ʼn hoë-vet dieët en water (HFD) of 'n kafeedieët groep wat die HFD met die byvoeging van 15% sukrose/fruktose in hul drink water (CFD) ontvang. Fastende glukose en insulien, binneaarse glukose toleransie toets (IVGTT), glukose gestimuleerde insulien sekresie tempo en 2-deoxi-[3H]-D-glukose opname in spier, lewer en vet is gebruik om die effek van die dieët op glukosemetabolisme te bepaal. Die pankreata is uitgehaal vir immunohistochemiese identifisering van β-selle (insulien), α-selle (glukagoon), GLUT2 (glukose transport) en MIB5 (proliferasie). Monsters van die pankreata was ook vir elektronmikroskopie versamel. Resultate en bespreking: Voeding van ʼn CFD aan Wistar rotte induseer vetsug, insulienweerstandigheid en glukose-intoleransie Teen twaalf maande toon die rotte 'n verswakte respons tot glukose met verhoogde IVGTT piekwaardes, AUC waardes en glukose opruimingswaardes. Terselfdetyd is die glukose gestimuleerde insuliensekresie tempo (GS-ISR) ook verswak. Gestimuleerde glukose opruiming, soos deur 2-deoxi-[3H]-D-glukose opname bepaal, was verlaag in spier en vetweefsel teen drie maande. Teen twaalf maande, weens die ouderdom van die rotte, is die gestimuleerde glukose opname verlaag in vergelyking met drie maande sonder 'n verskil tussen groepe. Na drie maande kon geen sigbare morfologiese verskille met ligmikroskopie tussen die diëte waargeneem word nie. Teen twaalf maande is morfologiese verskille waargeneem in beide die HFD en die CFD groepe. In die HFD groep is groot hipertrofiese onreëlmatige eilande met fibrotiese verandering waargeneem. In die CFD groep was die morfologiese verandering meer gevorder met fibrotiese onderverdeling en ontwrigting van die normale endokriene rangskikking. Die teenwoordigheid van inflammatoriese selle in die geaffekteerde eilande is verenigbaar met T2D. Afleiding: Die voer van 'n hoë-vet dieët aan Wistar rotte veroorsaak vetsug, abdominale adipositeit en insulienweerstandigheid. Die byvoeging van sukrose/ fruktose tot die hoë-vet dieët vererger die insulienweerstandigheid en veroorsaak glukoseintoleransie en matige hiperglukemie. Morfologiese veranderings in die groter eilande was verenigbaar met T2D.

Insulin resistance is an early marker of Type II diabetes. Excessive lipid accumulation in muscle and liver leads to insulin resistance, and lowering tissue lipids causes an enhancement of insulin action. The enzyme AMP-activated protein kinase (AMPK) is activated when cellular energy levels are compromised, such as during exercise; this enhances fuel oxidation and inhibits energy consuming processes. The hypothesis in this thesis was that activating AMPK in a lipid-induced insulin resistant state leads to tissue lipid reduction and improved insulin sensitivity. Insulin resistant high-fat fed (HF-) rats were administered 5-aminoimidazole-4-carboxamide-1-??-D-ribofuranoside (AICAR), a specific AMPK activator. During an euglycaemic hyperinsulinaemic clamp performed 24h later, HF-rats showed increased whole body, muscle and liver insulin action, independent of changes in PKB-phosphorylation. The liver had reduced triglycerides, malonyl-CoA and increased IkB-a content. A lowering of muscle malonyl-CoA was consistent with conditions favouring increased lipid utilisation. Normal, chow-fed rats also showed improved insulin action post-AICAR. Further studies showed that basal glucose uptake was not increased 24h after AICAR, suggesting that AMPK activation had caused an increase in insulin sensitivity. Diacylglycerols and triglycerides, but not ceramides, were reduced in the liver of AICAR treated HF-rats, suggesting lipid reduction as a likely mediator of enhanced liver insulin action. These lipid species were not reduced in muscle. AICAR administration to HF-rats lowered plasma glucose and fatty acids (FA) acutely, probably due to increased muscle glucose uptake and FA oxidation. Glycogen was reduced in liver and increased in muscle, suggesting glucose mobilisation from liver to muscle. Adrenergic blockade excluded the sympathetic nervous system in the acute AICAR effects. AMPK was activated in white muscle and liver of HF-rats immediately after AICAR, the same tissues that exhibited later improved insulin sensitivity. Tracer technologies used to investigate glucose and lipid fluxes showed that AMPK activation in white muscle simultaneously increased both glucose and FA uptake and their metabolism, with glucose also being stored as glycogen. The liver showed lower lipid synthesis, consistent with reduced liver lipid accumulation observed 24h post-AICAR. In conclusion, these results suggest that activation of AMPK leads to selective tissue lipid reduction and improved insulin action, and is a potential target for the treatment of insulin resistance and type II diabetes.

Insulin resistance is an early marker of Type II diabetes. Excessive lipid accumulation in muscle and liver leads to insulin resistance, and lowering tissue lipids causes an enhancement of insulin action. The enzyme AMP-activated protein kinase (AMPK) is activated when cellular energy levels are compromised, such as during exercise; this enhances fuel oxidation and inhibits energy consuming processes. The hypothesis in this thesis was that activating AMPK in a lipid-induced insulin resistant state leads to tissue lipid reduction and improved insulin sensitivity. Insulin resistant high-fat fed (HF-) rats were administered 5-aminoimidazole-4-carboxamide-1-??-D-ribofuranoside (AICAR), a specific AMPK activator. During an euglycaemic hyperinsulinaemic clamp performed 24h later, HF-rats showed increased whole body, muscle and liver insulin action, independent of changes in PKB-phosphorylation. The liver had reduced triglycerides, malonyl-CoA and increased IkB-a content. A lowering of muscle malonyl-CoA was consistent with conditions favouring increased lipid utilisation. Normal, chow-fed rats also showed improved insulin action post-AICAR. Further studies showed that basal glucose uptake was not increased 24h after AICAR, suggesting that AMPK activation had caused an increase in insulin sensitivity. Diacylglycerols and triglycerides, but not ceramides, were reduced in the liver of AICAR treated HF-rats, suggesting lipid reduction as a likely mediator of enhanced liver insulin action. These lipid species were not reduced in muscle. AICAR administration to HF-rats lowered plasma glucose and fatty acids (FA) acutely, probably due to increased muscle glucose uptake and FA oxidation. Glycogen was reduced in liver and increased in muscle, suggesting glucose mobilisation from liver to muscle. Adrenergic blockade excluded the sympathetic nervous system in the acute AICAR effects. AMPK was activated in white muscle and liver of HF-rats immediately after AICAR, the same tissues that exhibited later improved insulin sensitivity. Tracer technologies used to investigate glucose and lipid fluxes showed that AMPK activation in white muscle simultaneously increased both glucose and FA uptake and their metabolism, with glucose also being stored as glycogen. The liver showed lower lipid synthesis, consistent with reduced liver lipid accumulation observed 24h post-AICAR. In conclusion, these results suggest that activation of AMPK leads to selective tissue lipid reduction and improved insulin action, and is a potential target for the treatment of insulin resistance and type II diabetes.

The past two decades have seen an explosive increase of obesity rates worldwide, with more than one billion adults overweight and 300 million of them obese. Obesity and its associated complications have become leading causes of morbidity and mortality in the United States and major contributing factors to the rising costs of national health care. The pathophysiology of obesity and type 2 diabetes in rodents and humans is characterized by low-grade inflammation and chronic activation of immune pathways in adipose tissue and liver. The CD40 receptor and its ligand, CD40L, initiate immune cell signaling promoting inflammation, but conflicting data on CD40L-null mice confound its role in obesity-associated insulin resistance. A clear understanding of how CD40 and its ligand communicate to regulate and sustain the inflammatory environment of obesity is lacking. Here we demonstrate that CD40 receptor deficient mice on a high-fat diet display the expected decrease in hepatic cytokine levels, but paradoxically exhibit liver steatosis, insulin resistance and glucose intolerance compared with their age-matched wild-type controls. Hyperinsulinemic-euglycemic clamp studies also demonstrated insulin resistance in glucose utilization by the CD40-null mice compared with wild-type mice. In contrast to liver, visceral adipose tissue in CD40 deficient animals harbors elevated cytokine levels and infiltration of inflammatory cells, particularly macrophages and CD8+ effector T cells. In addition, ex vivo explants of epididymal adipose tissue from CD40-null mice display elevated basal and isoproterenol-stimulated lipolysis, suggesting a potential increase of lipid efflux from visceral fat to the liver. These findings reveal that 1) CD40-null mice represent an unusual model of hepatic steatosis with reduced hepatic inflammation, and 2) CD40 unexpectedly functions in adipose tissue to attenuate the chronic inflammation associated with obesity, thereby protecting against hepatic steatosis.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The cardiac isoform of acetyl-CoA carboxylase (ACCβ) produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid (FA) uptake; thus increased ACCβ activity decreases fatty acid utilization thereby potentially leading to intracellular myocardial lipid accumulation and insulin resistance (IR). Previous studies show that greater flux through the hexosamine biosynthetic pathway (HBP) contributes to the development of IR. In light of this, we hypothesize that increased HBP flux induces ACCβ gene expression thereby contributing to the onset of IR. Our initial work focused on ACCβ gene promoter regulation and suggest that the HBP modulates upstream stimulatory factor 2 (USF2) thereby inducing ACCβ gene expression. Here, we further investigated HBP-mediated regulation of ACCβ gene expression by transiently transfecting cardiac-derived H9c2 cells with an expression vector encoding the rate-limiting HBP enzyme (GFAT) ± the full length ACCβ and 4 truncated promoter-luciferase constructs, respectively. GFAT overexpression increased ACCβ gene promoter activity for the full length and 3 larger deletion constructs (p<0.001 vs. controls). However, GFAT-mediated and USF2-mediated ACCβ promoter induction was blunted when co-transfected with the -38/+65 deletion construct suggesting that USF2 binds to the proximal promoter region (near start codon). Further investigation proves that USF2 binds to ACCβ promoter and activates it, but that USF2 is not O-GlcNAc modified even though there is a strong correlation between increased O-GlcNac levels and USF2 activation of ACCβ. This would suggest that there is another O-GlcNac modified factor involved in this regulatory pathway. Our study demonstrates that increased HBP flux induces ACCβ gene promoter activity via HBP modulation of USF2. We propose that ACCβ induction reduces fatty acid oxidation, thereby leading to intracellular lipid accumulation (FA uptake>>FA oxidation) and the onset of cardiac IR. / AFRIKAANSE OPSOMMING: Die kardiale isoform van asetiel-CoA karboksilase (ACCβ) produseer maloniel-CoA, ‘n kragtige inhibeerder van mitochondriale vetsuur (VS) opname, en om hierdie rede sal verhoogde ACCβ aktiwiteit, vetsuur gebruik verlaag en potensieël aanleiding gee tot intrasellulêre miokardiale lipiedophoping en insulienweerstand (IW). Vorige studies toon dat groter fluks deur die heksosamienbiosintetiese weg (HBW) bydra tot die ontwikkeling van IW. In die lig hiervan hipotetiseer ons dat verhoogde HBW fluks, ACCβ geenuitdrukking induseer, en sodoende tot die onstaan van IW bydra. Ons aanvanglike werk het op ACCβ geenpromotorregulering gefokus, en voorgestel dat die HBW die opstroom stimuleringsfaktor 2 (USF2) moduleer en dus ACCβ geen uitdrukking induseer. Hier het ons verder die HBW-gemedieërde regulering van ACCβ-geenuitdrukking deur kortstondige tranfeksie van kardiaalverkrygde H9c2 selle met ‘n uitdrukkingsvektor wat kodeer vir die tempo-bepalende HBW ensiem (GFAT) ± die volle lengte ACCβ, en vier afgestompte promotor-lusiferase konstrukte onderskeidelik, te ondersoek. GFAT ooruidrukking het ACCβ geenpromotor aktiwiteit vir die volle lengte, en drie groter uitwissingskonstrukte verhoog (p<0.001 vs. kontrole). Hoewel GFAT- en USF2-gemedieërde ACCβ promotorinduksie tydens ko-transfeksie van die -38/+65 uitwissingskonstruk versag was, is dit voorgestel dat USF2 aan die proksimale promotor area (naby die beginkodon) bind. Verdere ondersoek bewys ook dat USF2 aan die ACCβ promotor bind en dit aktiveer, maar dat USF2 nie O-GlcNAc gemodifiseer word nie ten spyte van ‘n sterk korrelasie tussen verhoogde O-GlcNac vlakke en USF2 aktivering van ACCβ. Dit kan dus voogestel word dat daar ‘n alternatiewe O-GlcNac gemodifiseerde faktor betrokke is in hierdie reguleringsweg. Ons studie demonstreer dat verhoogde HBW fluks ACCβ geenpromotor aktiwiteit via HBW modulering van USF2 veroorsaak. Ons stel voor dat ACCβ induksie vetsuuroksidasie verlaag en so tot intrasellulêre lipiedophoping (VS opname >> VS oksidasie) en die onstaan van kardiale IW lei.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background - Although acute hyperglycemic episodes are linked to lower glucose uptake, underlying mechanisms driving this process remain unclear. We hypothesized that acute hyperglycemia triggers reactive oxygen species (ROS) production and increases non-oxidative glucose pathway (NOGP) activation, i.e. stimulation of advanced glycation end products (AGE), polyol pathway (PP), hexosamine biosynthetic pathway (HBP) and protein kinase C (PKC) activation. These mechanisms attenuate cellular function, and may indeed decrease insulin-mediated cardiac glucose uptake. The role of the pentose phosphate pathway (PPP) under high glucose/diabetic conditions is a subject of contention. Activation of the PPP enzyme transketolase (TK) (by benfotiamine/BFT or thiamine) reduces flux via the other four NOGPs, and is associated with beneficial outcomes. Our aim was therefore to evaluate the effects of acute hyperglycemia on insulin-mediated glucose uptake in a cardiac-derived cell line. Specifically, we aimed to elucidate the role of ROS and NOGP induction under these conditions. Methodology - H9c2 cardiomyoblasts were exposed to 25 mM glucose for 24 hr vs. 5.5 mM glucose controls ± modulating agents during last hour of glucose exposure: a) antioxidant #1 for mitochondrial ROS (250 μM 4-OHCA), b) antioxidant #2 for NADPH oxidase-generated ROS (100 μM DPI), c) NOGP inhibitors – 100 μM aminoguanidine (AGE), 5 μM chelerythrine (PKC); 40 μM DON (HBP); and 10 μM zopolrestat (PP). We also employed BFT (50 and 100 μM) in vitro, while the effects of in vivo thiamine administration were assessed in hearts of an obese/diabetic rat model of pre-diabetes and diabetes, the OLETF strain. We evaluated insulin sensitivity by glucose uptake assay (flow cytometry), GLUT4 translocation (transfection of HA-GLUT4-GFP construct) and protein kinase B (Akt) activity assay. ROS levels (mitochondrial, intracellular) were measured by flow cytometry analysis of specific fluorescent probes. Markers of each NOGP were also assessed. Results - Acute hyperglycemia elevated ROS, activated NOGPs and blunted glucose uptake. However, TK activity (marker of PPP) did not change. Respective 4-OHCA and DPI treatment blunted ROS production, diminished NOGP activation and normalized glucose uptake. NOGP inhibitory studies identified PKCβII as a key downstream player in lowering insulin-mediated glucose uptake. When we employed BFT (known to shunt flux away from NOGPs and into the PPP), it decreased ROS generation and NOGP activation, and restored glucose uptake under acute hyperglycemic conditions. In vivo thiamine administration reduced markers of the other NOGP, while it attenuated (mainly in the pre-diabetic phase) the metabolic dysfunction observed in the OLETF rats. Conclusions - This study demonstrates that acute hyperglycemia elicits a series of maladaptive events that function in tandem to reduce glucose uptake, and that antioxidant treatment and/or attenuation of NOGP activation (PKC, polyol pathway) may limit the onset of insulin resistance. / AFRIKAANSE OPSOMMING: Agtergrond – Alhoewel akute hiperglisemie voorvalle gekoppel is aan verlaagde glukose opname, is die onderliggende meganismes wat die proses dryf steeds onduidelik. Ons hipotetiseer dat akute hiperglisemie aanleiding gee tot die produksie van reaktiewe suurstofspesies (RSS) en toename in nie-oksidatiewe glukose weg (NOGW) aktivering, i.e. stimulering van gevorderde glukasie eindprodukte (GGE), poliolweg (PW), heksosamien biosintetiese weg (HBW) en proteïenkinase C (PKC) aktivering. Hierdie meganismes verminder sellulêre funksie, en mag inderdaad insulien-bemiddelde kardiêre glukose opname verlaag. Die rol van die pentosefosfaatweg (PFW) onder hoë glukose/diabetiese kondisies is ‘n onderwerp van stryd. Aktivering van die PFW ensiem transketolase (TK) (deur benfotiamien/BFT of tiamien) verminder fluks deur die ander vier NOGWë, en is geassosieer met voordelige uitkomste. Ons doel was dus om die effekte van akute hiperglisemie op insulien-bemiddelde glukose opname te evalueer in ‘n kardiaal-afkomstige sellyn. Meer bepaald het ons gepoog om die rol van RSS en NOGW induksie onder hierdie kondisies te verstaan. Metode – H9c2 kardiomioblaste is aan 25 mM glukose vir 24 h blootgestel vs. 5.5 mM glukose kontroles ± moduleeragente tydens die laaste uur van glukose blootstelling: a) anti-oksidant #1 vir mitochondriese RSS (250 μM 4-OHCA), b) anti-oksidant #2 vir NADPH oksidase-gegenereerde RSS (100 μM DPI), c) NOGW inhibeerders – 100 μM aminoguanidien (GGE), 5 μM cheleritrien (PKC); 40 μM DON (HBW); en 10 μM zopolrestaat (PW). Ons het ook BFT (50 en 100 μM) in vitro aangewend, terwyl die effek van in vivo tiamien aanwending geassesseer is in die harte van ‘n vetsugtige/diabetiese rotmodel van pre-diabetes en diabetes, die OLETF lyn. Ons het insuliensensitiwiteit deur ‘n glukose opname toets (vloeisitometrie), GLUT4 translokasie (transfeksie van HA-GLUT4-GFP konstruk) en proteïenkinase B (Akt) aktiwiteitstoets, geëvalueer. RSS vlakke (mitochondries, intrasellulêr) is gemeet deur vloeisitometriese analise van spesifieke fluoresserende peilers. Merkers van elke NOGW is ook geassesseer. Resultate - Akute hiperglisemie het RSS verhoog, NOGWë geaktiveer en glukose opname versag. TK aktiwiteit (merker van PFW) het egter nie verander nie. Onderskeidelike 4-OHCA en DPI behandeling het RSS produksie versag, NOGW aktivering verminder en glukose opname genormaliseer. NOGW onderdrukking studies het PKCβII geïdentifiseer as ‘n sleutel deelnemer in verlaging van insulien-bemiddelde glukose opname. Die aanwending van BFT (bekend vir die wegvoer van fluks vanaf NOGWë na die PFW), het RSS skepping en NOGW aktivering verlaag, en glukose opname herwin onder akute hiperglisemiese kondisies. In vivo tiamien toediening het merkers van die ander NOGW verlaag, terwyl dit die metaboliese disfunksie waargeneem in die OLETF rotte (hoofsaaklik in die pre-diabetiese fase) verminder het. Gevolgtrekking – Hierdie studie demonstreer dat akute hiperglisemie ‘n reeks van wanaangepaste voorvalle ontlok wat gesamentlik funksioneer om glukose opname te verlaag, en dat anti-oksidant behandeling en/of vermindering van NOGW aktivering (PKC, poliolweg), die aanvang van insulien weerstand mag beperk.

published_or_final_version / Zoology / Master / Master of Philosophy

Thesis (MScMedSc)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Obesity is a major risk factor for ischaemic heart disease. Obesity-induced metabolic abnormalities have been associated with increased oxidative stress which may play an important role in the increased susceptibility to myocardial dysfunction and ischaemiareperfusion (I/R) injury seen in obesity. The pineal gland hormone, melatonin, has powerful antioxidant properties. Previous studies have shown that short-term or acute melatonin administration protects the normal healthy heart of lean animals against I/R damage. However, the effects of melatonin on the heart in obesity remain unknown. Moreover, the myocardial signalling mechanisms associated with the cardioprotective effects of melatonin have not been established. Using a rat model of diet induced obesity, we set out to: 1) investigate the effects of chronic melatonin administration on the development of diet-induced systemic alterations including biometric and metabolic parameters and oxidative stress, 2) determine whether chronic melatonin treatment protects the myocardium against ischaemia-reperfusion injury, and 3) determine whether melatonin treatment confers cardioprotection by altering the reperfusion injury salvage kinase (RISK) pathway signalling and the pro-apoptotic p38 MAPK, AMPK and GLUT-4 expression. Male rats weighing 200±20g were randomly allocated to four groups: 1) C, control rats receiving a standard commercial rat chow and drinking water without melatonin; 2) CM, control rats receiving melatonin (4mg/kg/day) in drinking water; 3) D, diet-induced obesity rats, receiving a high calorie diet and drinking water without melatonin; 4) DM, diet-induced obesity rats, receiving melatonin in drinking water. After 16 weeks of treatment and feeding, rats were weighed and blood and myocardial tissue collected to document biochemical and molecular biological changes. Hearts were perfused on the isolated working rat heart perfusion apparatus for the evaluation of myocardial function and infarct size. The Reperfusion Injury Salvage Kinases (RISK) pathway (PKB/Akt (Ser-473), ERK p42/ p44) and p38 MAPK (mitogenactivated protein kinase) were investigated in pre-and post-ischaemic hearts using Western blotting techniques. Post-ischaemic activation of AMPK (5’AMP-activated protein kinase) (Thr- 172) and GLUT-4 (glucose transporter) expression were also investigated. Serum and baseline myocardial glutathione (GSH) content were measured. In addition, serum lipid peroxidation products: thiobarbituric reactive substances (TBARS), conjugated dienes (CD) and lipid hydroperoxide (LOOH), were also determined. The high-calorie diet caused increases in body weight, visceral adiposity, heart weight, serum insulin, leptin, blood triglycerides, and low HDL-cholesterol levels. Blood glucose levels were similar for both diet fed rats and controls. Myocardial glutathione, serum glutathione, total cholesterol, TBARS, LOOH, CD as well as total cholesterol (TC) levels were not affected by the high calorie diet. Chronic melatonin treatment reduced body weight gain, visceral adiposity, heart weight, blood triglycerides, serum insulin, HOMA index, serum leptin (DM vs D, p<0.01), and increased blood HDL-C in diet treated rats while there was no effect on these parameters in control rats, despite the reduction in body weight, heart weight and visceral adiposity. Melatonin treatment had no effect on myocardial or serum GSH and LOOH in either control or diet animals. It however reduced TBARS and CD in the diet and control groups, respectively. At baseline, chronic melatonin treatment caused a significant increase in phospho-PKB/total PKB ratio and a concomitant reduction in phospho-p38 MAPK/total p38 MAPK ratio of control hearts while there were no such effects on diet-induced-obesity hearts. Infarct size was significantly reduced by melatonin in both diet and control groups (DM: 16.6±2.0%; D: 38.4±2.6% (p < 0.001), and CM: 12.8±1.5%; C: 30.4±1.0%, p<0.001). After coronary artery occlusion and 30 minutes of reperfusion, melatonin increased percentage recovery of aortic output (DM: 28.5±6.5%; D: 6.2±6.2%, p<0.01), cardiac output (DM: 44.4±5.2%; D: 26.6±5.1%, p < 0.01) and total work (DM: 34.5±5.6%; D: 20.4±7.9%, p<0.05) of diet-induced obesity hearts, while having no effect on control hearts. During reperfusion, hearts from melatonin treated rats had increased activation of PKB/Akt (p<0.01), ERK42/44 (p<0.05), and reduced p38 MAPK activation (p<0.05). There was no difference in post-ischaemic activation of AMPK (Thr-172) and GLUT-4 expression in either control or diet fed rats. We successfully demonstrated that chronic melatonin treatment prevented the development of diet-induced metabolic abnormalities and improved ex vivo myocardial function. Melatonin protected the heart against ischaemia-reperfusion injury that was exacerbated in obesity. This was achieved by activation of the RISK pathway. The antioxidant properties of melatonin were involved in these cardioprotective effects. / AFRIKAANSE OPSOMMING: Vetsug of obesiteit is een van die hoof risikofaktore vir iskemiese hartsiekte. Obesiteitgeinduseerde metaboliese abnormaliteite gaan met verhoogde oksidatiewe stres gepaard wat op sy beurt ‘n belangrike rol mag speel in die miokardiale wanfunksie en verhoogde vatbaarheid vir iskemie-herperfusie (I/H) beskadiging, kenmerkend van vetsug. Melatonien, die hormoon afgeskei deur die pineaalklier, is ‘n kragtige anti-oksidant. Vorige studies het getoon dat kort-termyn of akute toediening van melatonien die normale hart van gesonde diere teen I/H beskadiging deur middel van sy anti-oksidant aksies beskerm. Die effek van melatonien op die hart in obesiteit is egter nog onbekend. Hierbenewens is die miokardiale seintransduksie meganismes geassosieer met die beskermende effekte van die hormoon nog nie ontrafel nie. ‘n Model van dieet-geinduseerde obesiteit in rotte is gebruik om die volgende te bepaal: (i) die effek van kroniese melatonientoediening op die ontwikkeling van dieet-geinduseerde sistemiese veranderinge soos biometriese en metaboliese parameters en oksidatiewe stres (ii) die effek van kroniese melatonienbehandeling op die respons van die hart op I/H beskadiging en (iii) die rol van herperfusie beskadiging op die aktivering van PKB/Akt en ERK42/44 (die sg RISK seintransduksiepad), die pro-apoptotiese p38MAPK, AMPK sowel as die uitdrukking van GLUT-4. Manlike Wistar rotte (200±20g) is ewekansig in vier groepe verdeel: (i) C, kontrole rotte wat ‘n standaard rotdieet en drinkwater sonder melatonien ontvang (ii) CM, kontrole rotte wat melatonien (4mg/kg/dag) ontvang (iii) D, dieet-geϊnduseerde vet rotte wat ‘n hoë kalorie dieet en drinkwater sonder melatonien ontvang (iv) DM, dieet-geϊnduseerde vet rotte wat melatonien (4mg/kg/dag) in die drinkwater ontvang. Na 16 weke van behandeling, is die rotte geweeg, bloed en hartweefsel gekollekteer vir biochemiese en molekulêre biologie bepalings. Harte is geperfuseer volgens die werkhartmodel, blootgestel aan iskemie/herperfusie vir evaluering van funksionele herstel en infarktgrootte. Uitdrukking en aktivering van PKB/Akt (Ser-473), ERKp42/p44 en p38MAPK van pre-en postiskemiese hartweefsel is met behulp van Western blot bepaal. Postiskemiese aktivering van AMPK (5’AMP-aktiveerde proteϊen kinase) (Thr-172) en GLUT-4 (glukose transporter) is op soortgelyke wyse bepaal. Serum en basislyn hartweefsel glutatioon (GSH) inhoud asook tiobarbituursuur reaktiewe substans (TBARS), gekonjugeerde diene (CD) en lipiedhidroperoksied (LOOH) konsentrasies is bepaal. Resultate Die hoë kalorie diet het ‘n toename in liggaamsgewig, visserale vet, hartgewig, serum insulien, leptien, plasma trigliseried en lae HDL-cholesterol vlakke teweegebring. Bloed glukosevlakke was egter dieselfde in die vet en kontrole rotte. Miokardiale glutatioon, serum glutatioon, totale cholesterol, TBARS, LOOH, CD is nie deur die dieet beinvloed nie. Chroniese melatonien behandeling het die liggaamsgewig, visserale vet, hartgewig, plasma trigliseried, serum insulien en leptien, HOMA indeks verlaag (DM vs D, p<0.05) en die HDL-cholesterol verhoog in die dieetrotte, terwyl dit geen effek op hierdie parameters in kontrole rotte gehad het nie (uitgesonderd ‘n afname in liggaamsgewig, hartgewig en visserale vet). Melatonien behandeling het geen effek op hart of serum GSH en LOOH in kontrole en vet rotte gehad nie. Dit het egter die TBARS en CD in beide vet en kontrole rotte verlaag. Chroniese melatonien toediening het ‘n beduidende toename in basislyn fosfo PKB//totale PKB ratio en ‘n afname in fosfo p38MAPK/totale p38MAPK ratio teweegebring in harte van kontrole rotte, maar soortgelyke effekte is nie in die harte van die vet rotte waargeneem nie. Infarktgrootte is beduidend deur melatonienbehandeling verlaag in beide dieet en kontrole groepe (DM: 16.6± 5.2%, D: 38.4 ±2.6% (p<0.001); CM: 12.8± 1.5%; C 30.4±1.0 (p<0.001). Na koronere arterie afbinding en 30 min van herperfusie, het melatonien die persentasie herstel van aorta omset (DM: 28.5± 6.5%; D: 6.2± 6.2%, p<0.01), kardiale omset ( DM: 44.4± 5.2%D: 26.6±5.1%, p<0.01) en totale werk (DM: 34.5 5.6%; D 20.4± 7.9%, p<0.05) in die harte van dieetrotte verbeter, terwyl dit sonder effek was in kontrole harte. Tydens herperfusie het harte van melatonienbehandelde rotte verhoogde aktivering van PKB/Akt (p<0.01) en ERKp42/p44 (p<0.05) getoon, terwyl aktivering van p38MAPK verlaag is (p<0.05). Geen verskil in postiskemiese aktivering van AMPK en GLUT-4 uitdrukking is in beide kontrole en dieetrotte waargeneem nie. Ons het daarin geslaag om aan te toon dat chroniese melatonienbehandeling die ontwikkeling van dieet-geϊnduseerde metaboliese abnormaliteite beduidend kan voorkom en ex vivo miokardiale funksie verbeter. Melatonien het ook die hart teen iskemie/herperfusie beskadiging beskerm in beide kontrole en dieetrotte. Bogenoemde veranderinge het met aktivering van PKB/Akt en ERKp42/p44 gepaard gegaan. Die anti-oksidant effekte van melatonien was heelwaarskynlik hierby betrokke.