Laminitis is a crippling disease of horses that can result in chronic lameness and debilitation, and sometimes warrants euthanasia. It is a complication of inflammatory conditions such as gastrointestinal disease, and also occurs in obese, insulin-resistant horses with Equine Metabolic Syndrome (EMS). Inflammation and insulin resistance are risk factors for laminitis, and these mechanisms might converge to induce laminitis in susceptible animals. Systemic inflammation is often attributed to endotoxemia, although circulating endotoxin concentrations are not commonly measured in the clinical setting. Although a theoretic basis exists for endotoxemia in the pathogenesis of laminitis, administration of endotoxin alone does not induce the condition. This could be related to differences between experimental models and naturally occurring disease. Studies presented in this dissertation address the overall hypothesis that systemic inflammation causes laminitis and new experimental models can be developed to better represent clinical disease. Associations between systemic inflammation and laminitis were first established by measuring blood inflammatory cytokine expression during a laminitis induction model. A clinically relevant endotoxin model that induced laminitis was then sought, but endotoxin administration alone was insufficient to cause laminitis and endotoxin tolerance developed. Endotoxemia was therefore evaluated in conjunction with predisposing factors such as obesity. In horses with EMS, endotoxin infusion caused exaggerated inflammatory responses, and derangements in glucose homeostasis were more pronounced. Laminitis, however, did not develop. Repeated inflammatory events are implicated in the pathogenesis of sepsis-associated organ failure, so a final study was performed to test whether preexisting endotoxemia increased the risk of laminitis during subsequent carbohydrate overload-induced systemic inflammation. This did not occur, however systemic inflammation was more pronounced in horses that developed laminitis compared to non-responders, and tissues rather than circulating leukocytes appeared to be the major source of inflammatory mediators. Our results do not support a role for endotoxin as the causal agent of laminitis, even when combined with predisposing factors. Tissues appear to be an important source of inflammatory mediators, therefore their role in laminitis should be further characterized. Additionally, future investigations should determine whether exaggerated inflammatory responses and loss of glycemic control increase the risk of laminitis in horses with EMS.

Intestinally derived chylomicron remnants (CM-r) may contribute to atherogenic dyslipidemia during the Metabolic Syndrome (Mets). However, the combined effects of ezetimibe (EZ) and simvastatin (SV) on post-prandial (PP) dyslipidemia during MetS remains unclear, nor is it known whether the combination has a synergistic anti-atherogenic effect on CM-r arterial retention. The first objective was to delineate the effects of EZ+SV therapy on intestinal cholesterol flux and CM PP metabolism in the JCR:LA-cp rat, a model of MetS. The second objective was to quantify the impact of EZ+SV therapy on arterial retention of CM-r and subsequent myocardial lesion development in the JCR:LA-cp rat. EZ+SV therapy decreased net intestinal cholesterol absorption in MetS rats. Furthermore, EZ+SV therapy reduced arterial retention of CM-r and frequency of myocardial lesions in MetS rats. In conclusion, EZ+SV therapy reduces arterial retention of CM-r and myocardial lesion development possibly through its beneficial effects on cholesterol transport and PP-metabolism. / Nutrition and Metabolism

Trans-11 vaccenic acid (VA) is the predominant trans fatty acid in dairy fat and is the major precursor to endogenous synthesis of cis9,trans11-conjugated linoleic acid (CLA) in humans and animals. Epidemiological studies have shown the positive association between trans fat intake and incidence of coronary heart disease. Nevertheless, CLA, categorized as a group of trans fatty acids, has been shown to possess anti-carcinogenic, hypolipidemic and anti-diabetic benefits in several animal models as well as certain human populations, possibly via activating peroxisome proliferator-activated receptor (PPAR) related metabolic pathways. The subsequent effort in enriching CLA in dairy products (e.g. butter) has led to a concomitant increase in VA, whose bioactivity and health implications were not fully appreciated. Interestingly, VA is the major natural trans fat found in the diet. Therefore, the objectives of this thesis were to assess the effect of dietary supplementation of synthetic VA on lipid metabolism especially during conditions of dyslipidemia and metabolic syndrome, and to delineate the intestinal and hepatic metabolic pathways potentially modulated by VA. The JCR:LA-cp rat model, when homozygous for the cp trait (cp/cp), develop leptin receptor deficiency which leads to symptoms of metabolic syndrome and pre-diabetes including obesity, insulin resistance, hepatic steatosis, hypertriglyceridemia and exacerbated production of hepatic very low-density lipoproteins and intestinal chylomicrons (CM). Gas chromatography analysis on nascent lymph shows that VA was effectively absorbed into the intestine. In addition, VA from natural source (i.e. beef fat) showed higher intestinal bioavailability compared to synthetic VA. Dietary supplementation of 1.0% (w/w) synthetic VA to JCR:LA-cp rats (but not lean healthy controls) demonstrated a profound reduction in plasma triglyceride, total cholesterol, low-density lipoprotein-cholesterol, non-esterified fatty acid and haptoglobin concentrations (51%, p<0.001; 40%, p<0.001; 50%, p<0.05; 20%, p<0.05 and 50%, p<0.001; respectively), as well as improvement in hepatic steatosis and postprandial lipaemia. Gastric infusion of VA also resulted in an acute reduction in CM secretion in response to a fat load (p<0.05). We also found that the overall hypolipidemic benefits of VA might be partially contributed by suppression of hepatic de novo lipogenesis, activation of PPAR- activity as well as up-regulation of PPAR- and PPAR- expression in the intestine. In conclusion, VA as a natural trans fat, possesses beneficial properties in a rat model of dyslipidemia and metabolic syndrome, suggesting potential for the prevention of cardiovascular disease risk. / Nutrition and Metabolism

Activation of inducible nitric oxide synthase (iNOS) and oxidative stress have been shown to be associated with compromised cardiovascular function in streptozotocin (STZ)-induced type 1 diabetes. The aim of the project is to investigate cardiovascular abnormalities in a rat model of type 2 diabetes (Zucker diabetes fatty or ZDF rats) and two models of metabolic syndrome (fructose-fed rats and Zucker obese rats), and to provide direct evidence linking iNOS and oxidative stress to abnormal cardiovascular function in these disorders. Blood pressure, cardiac contractility, cardiac index, regional flow, vascular resistance and venous tone were measured in diseased as well as normal rats. Biochemical analyses such as activities of iNOS, immunostaining of iNOS and western-blot analysis of iNOS in the heart tissue were carried out. The results showed that cardiac contractile response to dobutamine was compromised in the ZDF rats, and this was associated with increased myocardial protein expression as well as activity of iNOS. The formation of peroxynitrite was increased in the heart tissue of the ZDF rats. Selective inhibition of iNOS by 1400W (N-3-aminomethyl-benzyl-acetamidine) did not alter responses to dobutamine in the control rats, but augmented the contractile effects of dobutamine in the diabetic rats. The regional blood flow was altered in the ZDF rats, and iNOS played a negligible role in regulating regional flow in the ZDF rats. Although venous response to noradrenaline was also altered in the Zucker obese rats, NOS may not be involved in venous tone regulation. Anti-oxidative treatment with N-acetylcysteine inhibited the development of insulin resistance, blood pressure elevation and the increase of 8-isoprostane formation in the fructose-fed rats. We conclude that heart function is compromised and regional blood flow is altered in the ZDF rats. Activation of iNOS plays an important role in suppressing heart dysfunction but does not affect regional blood flow. In Zucker obese rats with metabolic syndrome, iNOS may not be involved in changes of venous function. Oxidative stress is associated with both abnormality of heart dysfunction in type 2 diabetes (by formation of peroxynitrite due to iNOS activation) and development of hypertension and insulin resistance in metabolic syndrome.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

The insulin resistance syndrome [INSR] is associated with increased cardiovascular risk and affects up to 25% of the Australian population. The mechanism underlying the relationship between the INSR and increased cardiovascular risk is controversial. We postulated that perturbations in the renin-angiotensin system [RAS] and endothelium-derived NO may be implicated in the development of early vascular changes in the INSR. Repeated measurements of arterial stiffness [using digital photoplethysmography] and haemodynamic parameters in response to vasoactive medications were used to demonstrate the functional expression of angiotensin II [Ang II] receptors and NO synthase [NOS]. Ang II acts via two main receptor sub-types: the Ang II type 1 [AT1] and Ang II type 2 [AT2] receptors. The AT1 receptor is central to the development of arterial stiffness and endothelial dysfunction. The role of AT2 receptors in humans is controversial but is postulated to counter-act AT1 receptor mediated effects in diseased vascular beds. We demonstrated increased AT1 and AT2 receptor-mediated effects in small to medium-sized arteries of subjects with early INSR [Chapter 6]. In addition, functional expression of AT2 receptors in adult insulin resistant humans [Chapter 5], but not in healthy volunteers [Chapter 4] was demonstrated. AT1 receptor blockade in subjects with early INSR resulted in improvements in vascular function, with a consequent functional down-regulation of AT2 receptors [Chapter 7]. Functional NOS expression was demonstrated to be increased in subjects with early INSR compared with healthy controls [Chapter 6]. This was postulated to be a homeostatic response to counteract early vascular changes in subjects with early INSR. AT1 receptor blockade in these subjects reduced functional NOS expression [Chapter 8]. In conclusion, patients with early INSR represent a model of early disease where early intervention may be able to reverse the process incited by the initial exposure to multiple cardiovascular risk factors. Early vascular changes in these individuals are mediated at least in part, by increased AT1 receptor activity and/or expression, and may be detected by changes in arterial stiffness indices and non-invasive vascular reactivity studies. There is a compensatory increase in AT2 receptor and NOS expression/activity to counter-act these vascular changes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.