Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) has emerged over the past decade as an important regulator of plasma cholesterol and cardiovascular disease risk. PCSK9 promotes degradation of low density lipoprotein (LDL) receptors, thereby decreasing LDL clearance. Accordingly, patients with gain-of-function mutations in PCSK9 have increased LDL cholesterol and increased risk of cardiovascular disease. Conversely, PCSK9 inhibitors recently approved by the FDA are effective in reducing LDL cholesterol. While the contribution of PCSK9 to familial hypercholesterolemia is well-established, less is understood about the role of PCSK9 in diseases in which hyperlipidemia results secondary to an initial disease insult. Hormonal regulation of PCSK9 is also incompletely understood. Understanding the regulators of PCSK9 and specific diseases in which it contributes to hypercholesterolemia is important for identifying additional mechanisms via which PCSK9 can be manipulated, and for choosing patient populations in which PCSK9 inhibitors will be effective. Here, we investigate PCSK9 in two diseases of secondary hyperlipidemia. In nephrotic syndrome, damage to kidney podocytes causes extreme proteinuria and hypercholesterolemia of unclear etiology. We show that plasma cholesterol and PCSK9 are dramatically elevated in mice made nephrotic by nephrotoxic serum treatment or podocyte apoptosis. Moreover, knockout of Pcsk9 protects mice from the effects of nephrotic syndrome on plasma lipids, particularly increased LDL cholesterol. Similarly, nephrotic patients show decreased plasma PCSK9 and cholesterol upon disease remission. Second, loss of adipose tissue in lipodystrophy results in low levels of the hormone leptin. Treatment of lipodystrophic patients with leptin reduces LDL cholesterol through unknown mechanisms. We used this background of hypoleptinemia to investigate the effects of leptin on PCSK9. We found that in female lipodystrophic patients, leptin treatment reduced plasma PCSK9, correlating with decreased LDL cholesterol. Similarly, in male leptin-deficient ob/ob mice, leptin also decreased plasma PCSK9 but did not affect plasma lipids. Our data show that PCSK9 is a novel regulator of hypercholesterolemia in nephrotic syndrome, suggesting that PCSK9 inhibitors may be an important therapy for this patient population with ambiguous treatment options. They also show that leptin can suppress PCSK9 expression, which may explain the observed decreases in LDL cholesterol upon leptin treatment of lipodystrophic patients. / Medical Sciences

Purpose. Study #1 examined the responses to facial immersion under states of altered cardiac filling. Study #2 sought to examine changes in postexercise hemodynamics and skin blood flow (SkBF) when lower blood pooling was manipulated using lower body pressure. Methods. Study #1---Six male subjects participated. They performed 30-s apneic facial immersions under: LBNP, LBPP, during post-exercise hypotension (PEH); & Control. MAP, HR, and SkBF were measured. Study #2---Subjects were exposed to LBNP, LBPP; or no pressure after 15-mins of cycle exercise at 70% of VO2 peak. HR, CO, SV, MAP, TPR, & SkBF, skin and esophageal temperature were recorded. Conclusions. Study #1---Cardiac parasympathetic response during facial immersion can be attenuated when cardiac filling is compromised. Study #2---(1) LBPP accelerates recovery of baseline hemodynamics while LBNP exacerbates the postexercise hemodynamic state relative to control. (2) Altering postexercise hemodynamics via LBPP may affect thermal responses.

Much remains unknown about the physiological effects of prolonged dietary carbohydrates on carnivorous fish such as rainbow trout, Oncorhynchus mykiss. In this thesis, the long-term effects of carbohydrates on trout glucose regulation are investigated by characterizing the metabolite profiles of fish fed with or without carbohydrates. Two groups of fish were maintained on either carbohydrate-rich (HC, 32.9% of diet by weight) or deficient (LC, 1% of diet by weight) diets for a six-month period. Plasma, liver and muscle tissue samples were assayed for various metabolites. Intravenous glucose tolerance tests were performed in each group and showed no significant differences in glucose tolerance. Continuous tracer infusion was used to measure glucose turnover in each group to determine whether changes in glucose concentration in response to a glucose load were due to changes in rates of glucose appearance (Ra), disappearance (Rd) or both. This research has demonstrated that rainbow trout can acclimate to high carbohydrate diets and modify several physiological parameters and biochemical pathways to both utilize and store newly available energy provided in the form of dietary glucose. (Abstract shortened by UMI.)

Adiponectin is a protein secreted exclusively from adipose tissue, which is speculated to increase acute fatty acid oxidation rates. High-intensity exercise has been reported to have a greater capacity to increase absolute fat oxidation rates, as compared to low-intensity exercise. Hence, the objective of this study was to investigate the acute effects of exercise intensity on adiponectin levels in young healthy women. Nine healthy, moderately active women (age = 22 +/- 2 years, BMI = 22.1 +/- 21.5 kg/m 2; VO2peak = 43.9 +/- 4.0 ml O2/kg/min) performed in a randomized order three experimental sessions: control (C) with no exercise and two equicaloric (350 kcal), low-intensity (LIE) and moderate-to-high-intensity (M-HIE) exercise sessions on a treadmill at 40% and 70% VO2peak, respectively. Experimental sessions took place during the follicular phase of the participant's menstrual cycle; therefore, sessions were spread out by at least one-month. Plasma adiponectin levels were measured before, at 15-minutes, at 30-minutes, and post exercise session (LI/M-HI) or control session. The results indicated that plasma adiponectin levels were comparable across conditions and remained unchanged over time within each session. These findings suggest that plasma adiponectin levels do not seem to be acutely modulated during and immediately after exercise of varying intensity in young healthy women.

The mineralocorticoid receptor (MR) was recently cloned from two fish species, Haplochromis burtoni (H. burtoni; hbMR) and rainbow trout (Oncorhynchus mykiss; rtMR). The objectives of the present study were to detect MR protein expression in rainbow trout ionoregulatory tissues, namely the gill and kidney, as well as to use the mammalian MR agonist aldosterone to selectively target and activate the rainbow trout MR to observe its effects on renal and branchial ionic regulation. Through immunohistochemistry and Western blot analysis using a heterologous antibody, MR protein expression was detected at the gill. Immunohistochemical analysis and flow cytometry data indicated that there was a high degree of colocalization of the rtMR to Na+-K+-ATPase rich cells at the gill, supporting the hypothesis that the rtMR is involved in branchial ionic regulation. Additionally, acute aldosterone treatment, but not cortisol treatment, stimulated a decrease in urine flow rate (UFR) and urinary sodium excretion at 1.5 h post-injection. The decrease in UFR occurred without any change in glomerular filtration rate (GFR), suggesting that the aldosterone-induced decrease in UFR was the result of increased tubular reabsorption of water, which in turn was presumably stimulated by enhanced Na+ reabsorption. The results of this study support the hypothesis that the rtMR is involved in renal and branchial ionic regulation and help to clarify the mechanisms through which corticosteroid-mediated ionic regulation occurs in euryhaline teleosts.

alpha1-Adrenoceptors (alpha1-ARs) are members of the G-protein coupled receptor superfamily of membrane proteins that mediate the actions of the endogenous catecholamines, adrenaline and noradrenaline. alpha 1-ARs are the prime mediators of vascular smooth muscle contraction and hypertrophic growth, and play a key role in the regulation of arterial blood pressure. Four weeks of high-salt diet (11% NaCl) significantly increased blood pressure (37.1%). An average two-fold decrease in alpha1D-AR mRNA expression was observed in the DA, ABA and EBA in high-salt diet fed trout. The chronically hypertensive fish exhibited a blunted vascular response to hypercapnia and exogenous catecholamines. These results suggest either a lowered vascular response capacity or that alpha1D-AR down-regulation occurred in high-salt diet fed fish as a consequence of the elevated blood pressure. The alpha1D-AR is, at least in part, involved in salt-induced high blood pressure in rainbow trout. This study is the first to characterize the alpha1-AR gene family at molecular level in any fish species, and provides the first evidence for a link between alpha1-ARs and salt-induced high blood pressure.

The purpose of this study was to gain an understanding of the success elements of elite big mountain (BM) freeskiers to perform their best while immersed in their challenging sport. Big Mountain freeskiing is a high risk alternative sport that takes place in an unpredictable mountain environment. Nine of the best BM freeskiers in the world participated in the study, seven men, and two women. A qualitative research design was used with a post-positivist paradigm. Face-to-face interviews were used to gather the data. The success elements that emerged from the interviews were categorized within three temporal categories; pre-performance preparation (line selection, visualization, and calmness), performance execution (confidence and focus), and post-performance evaluation (reflection, lessons learned, mindset). Two additional success elements were evident for all athletes interviewed; love for their sport and what they were doing, and a fully focused connection to that in which they were engaged. These latter two success elements were not confined by temporal boundaries and were presented separately due to their uniqueness and importance. These athletes carried an intensely focused connection to what they were doing, and had the ability to remain calm while facing challenging situations. The ability of these athletes to transfer certain success elements they employed in BM freeskiing to the rest of their lives was interesting and one that warrants further research.

A number of social and behavioural traits inherent to the individual are known to influence the outcome of social interactions in salmonid fish. In addition, recent work has raised the possibility that the prior physiological condition of a fish may predetermine its social status. Therefore, the hypothesis that elevated plasma cortisol levels influence social rank was tested, with the prediction that rainbow trout (Oncorhynchus mykiss) treated with cortisol would be relegated to subordinate social status in pairwise contests with an untreated conspecific. Experimental elevation of plasma cortisol significantly increased the probability that the treated fish within each pair became subordinate, an effect later identified as cortisol specific, as it was abolished by simultaneous treatment with the glucocorticoid receptor antagonist, RU486. One possible mechanism through which cortisol could exert this effect was subsequently investigated, namely that cortisol influenced social status through behavioural modifications mediated by changes in brain monoaminergic activity. (Abstract shortened by UMI.)

The identification of a multi-potent stem cell-like population (SP) within the adult murine heart infers new methods of cardiac repair, i.e. stem cells compensate for damage by generating new cardiomyocytes. Several studies have emphasized the dominance of the tissue microenvironment on the differentiation & functional properties of stem cells. Of particular interest was Cardiotrophin-1 (CT-1), a member of the Interleukin-6 (IL-6) family of cytokines. To assess whether CT-1 functioned as an activator of cardiac SP cells in the murine heart, an adenovirus containing the full length CT-1 driven by a ubiquitous promoter was generated. The adenovirus was administered via intra-cardiac injections and the effects of CT-1 were primarily assessed using fluorescence-activated cell sorting (FACS) analysis. Analysis showed a temporary increase in the cardiac SP of CT-1 injected hearts. A closer look at the SP cells in other tissues (liver, skeletal muscle and bone marrow) demonstrated that this phenomenon was cardiac specific. Interestingly the cardiac SP expressed the Leukemia Inhibitory Factor (LIF) receptor, which is required for CT-1 signaling through the 130 pathway. Protein analysis also showed that STAT3, a downstream member of the 130 pathway becomes activated in the heart during CT-1 injections. Preliminary results from co-culture experiments suggested that this increase was also accompanied by SP cell differentiation. These results proposed that CT-1 not only increased the cardiac SP size but may have also activated a cardiac differentiation program. The existence of a potential biologic stimulant (CT-1) for cardiac stem cells is an exciting prospect and offers support to the notion that cardiac repair may become a viable therapeutic option in the not too distant future.

The m196 mutation, a result of an ENU mutagenesis screen in zebrafish, disrupts embryonic development resulting in a delay of proper gill arch cartilage differentiation and a disruption of median and pectoral fin fold development. Mutant phenotypes arise at 24 hours post fertilization (hpf) with the onset of fin fold development and lead to early mortality between 30 hpf and 2 weeks from unknown causes. m196 delay and disruption of craniofacial chondrogenesis is possibly due to the initial displacement of the pharyngeal pouches and dismorphogenesis of the branchial arches. Cells expressing genetic markers of the fin fold and neural crest accumulate in a disorganized manner along the tail. Using meiotic mapping techniques, we have mapped the m196 mutation to a SSLP marker zCtg37A on LG17. We are analyzing candidate genes in search of the m196 mutation. These results will lead to the identification of a genetic factor essential for craniofacial and fin development in zebrafish.