One third of all diabetic patients develop kidney disease, or diabetic nephropathy. An important site of progressive injury in diabetic nephropathy is the glomerulus. Angiotensin II (Ang II) has been implicated as a key mediator in the progression of diabetic nephropathy. Ang II influences renal hemodynamics and modulates renal transport and growth. The intrarenal renin-angiotensin system (RAS) is responsible for local production of Ang II, independent of the systemic RAS. It is not known whether the glomerular RAS is involved in altered local Ang II production. Moreover, a number of studies have suggested downregulation of Ang II receptors in early diabetes, primarily attributed to the angiotensin type I (AT1) receptors. These studies determined the effect of early diabetes on the expression of components of the glomerular RAS, and on the status of a novel Ang II receptor, Ang II type 2 (AT 2) receptor. Three groups of rats were studied after two weeks: (1) control [C], (2) streptozotocin (STZ)-induced diabetes, with daily insulin to prevent ketosis but maintain hyperglycemia [D], and (3) STZ-induced diabetes, with normoglycemia maintained by insulin implants [D+I]. D rats had increased plasma glucose levels [C: 9.63 +/- 0.19 mM vs D: 37.79 +/- 1.63 mM (p < 0.001 vs C) vs D+I: 4.88 +/- 0.43 mM (p < 0.05 vs C); n = 12] and experienced renal hypertrophy, and a decrease in body weight compared to C and D+I rats. Plasma renin activity (PRA) was decreased in D but not significantly compared to C and D+I rats. Glomerular suspensions were isolated by sequential sieving after density gradient centrifugation. By competitive RT-PCR, D had no significant effect on glomerular mRNA expression of renin [C: 2,497.50 +/- 405 .03 vs D: 3,155.50 +/- 417.26 vs D+I: 2,490.00 +/- 645.80 fg mRNA/62.5 ng RNA; p = NS; n = 6], angiotensinogen (n = 4), or angiotensin converting enzyme (ACE: n = 5). By Western analysis, glomerular AT1 receptor protein expression was increased in D rats (341 +/- 127% of C; p < 0.05; n = 7), an effect partly reversed in D+I. By RT-PCR, AT2 receptor mRNA was increased in the cortex of D rats [C: 58,430 +/- 6,004 vs D: 83,675 +/- 3,575 vs D+I: 56,326 +/- 3,011 arbitrary units, (p < 0.005, D vs C and D+I); n = 6--7]. (Abstract shortened by UMI.)

This study tests the hypothesis that glucose tolerance in fish is related to nutrient preference and that this tolerance is correlated with hexokinase activity and tissue glucose transporters. Differences in glucose clearance abilities of three fish species, the carnivorous rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata) and the omnivorous bullhead catfish (Ameiurus melas) were investigated in this study. Glucose tolerance was compared between the three species and between feeding and 6 month fasted eels. Glucose tolerance was assessed by administering an intravenous glucose tolerance test (IGTT), injecting 250 mg glucose/kg body weight and taking blood samples over 24 h. Feeding eel and catfish were found to be more glucose tolerant than fasting eel and rainbow trout. Glucose turnover was investigated in the rainbow trout during the glucose tolerance test and the rate of appearance of glucose did not decrease until 2 h after a glucose load which may contribute to the glucose intolerance seen. To test what factors might contribute to differences in glucose tolerance, two steps within the glucose uptake pathway were examined in white muscle. (Abstract shortened by UMI.)

The AChE gene encodes three mRNAs; R, H and T differing in their choice of C-terminal exons. Of these, AChET is exclusively accumulated in the synaptic compartment of adult skeletal muscle fibres. The mechanisms underlying the tissue-specific expression of the T forms as well the targeting of AChE to the neuromuscular junction have yet to be elucidated. To this end, we examined factors influencing the expression of AChE by transfection of C2C12 muscle cells and direct gene transfer in rodent hindlimb muscles. Transfection of C2C12 muscle cells with cDNAs encoding H and T resulted in high levels of enzyme activity. The H non-muscle subunit was preferentially expressed, several-fold more than the T subunit. Furthermore, transfection of myoblasts with a cDNA containing all the sequences necessary for expression of all AChE splice variants resulted only in expression of T subunits. This indicated that selective expression in muscle fibres is due to exclusive splicing of T mRNA from the primary transcript. (Abstract shortened by UMI.)

Increased exposure to solar radiation in the UV-B range (280-320 nm) is occurring globally at least partly due to anthropogenic degradation of stratospheric ozone. The direct impact of present ambient (1.95 W/m$\sp2$) and sub-ambient doses of UV-B radiation on muscle and skin tissue antioxidants in mature zebrafish (Brachydanio rerio) and on their hatching success and survival in early life stages was measured. Exposure of mature fish to sub-ambient doses of UV-B radiation both in the presence (1.28 W/m$\sp2$) and absence (1.72 W/m$\sp2$) of a cellulose acetate filter (blocks all wavelengths below 290 nm) resulted in a significant depression in muscle/skin total glutathione (TGSH) levels compared with controls and low UV-B treated fish. Exposure to an ambient intensity of UV-B resulted in a significant decrease in muscle/skin glutathione peroxidase (GPx) activity compared with controls and low treated fish. A coincident significant increase in catalase activity compared with controls and low UV-B treated fish was also observed. Changes in SOD, catalase, GPx and GSH status suggests a UV-B-mediated increase in cytosolic superoxide anion radicals (O$\sb2\sp-$) and hydrogen peroxide. Newly fertilized eggs exposed to ambient UV-B for the duration of the hatching period exhibited 1.68% hatching success. Newly hatched larvae were observed to be more sensitive to UV-B than the adults. (Abstract shortened by UMI.)

This thesis examines the theory that rainbow trout (Oncorhynchus mykiss) regulate sodium through an epithelial sodium channel (ENaC) and a proton-ATPase. An attempt was made to clone an ENaC from a rainbow trout cDNA library and from total RNA through nested primers and PCR and RT-PCR resulting in RAPD-type E. coli DNA amplification and amplification of trout collagen, however, producing nothing related to ENaC. The second part focuses on the trout proton-ATPase B-subunit (H +-ATPase) that was used in a Northern hybridization tissue distribution. Phylogenetic analysis showed that the H+-ATPase is highly similar to other known B-subunits, particularly the brain isoform. H+-ATPase mRNA quantification attempts to examine the effect of various environmental factors failed due to the use of an improper internal control. Cortisol RIA's showed no change in levels in fish exposed to hypercapnia or reduced ion water but were significantly increased in the cortisol implanted fish.

In situ and in vivo experiments were performed on rainbow trout (Oncorhynchus mykiss) to examine (i) the direct effect of CO2 on the systemic vasculature and (ii) the influence of internal versus external hypercarbic acidosis on a variety of cardiovascular variables. Adult Atlantic salmon (Salmo salar) were exposed to acute, localized environmental hypercarbia (2.0 and 4.0% CO2 in air) over the gills via injections (60 ml/kg) of equilibrated seawater directly into the buccal cavity. Adult Atlantic salmon (Salmo salar) and Pacific spiny dogfish (Squalus acanthias) were exposed to acute environmental hypercarbia (approximately 20 min). Experiments were performed to examine the influence of environmental hypercarbia on aspects of cardiorespiratory physiology, and in separate series of experiments the muscarinic antagonist atropine (100 nmol kg-1; both species) and complete branchial denervation (dogfish) were used to investigate putative CO2-chemoreceptive sites on the gills and their link to the autonomic nervous system. The homogeneity of the cardiovascular and ventilatory responses amongst fish to elevated ambient CO2 was investigated by exposing six species of fish to acute environmental hypercarbia (approximately 20 min). The experiments were performed in vivo using two marine teleosts, Atlantic salmon (Salmo salar) and Pacific sanddab (Citharychthus sordidus); two freshwater teleosts, brown bullhead (Ictalurus nebulosus) and American eel (Anguilla rostrata); as well as one marine elasmobranch, the Pacific spiny dogfish (Squalus acanthias). (Abstract shortened by UMI.)

The total functional surface area of the gills is a key component in gas exchange and any reduction would predictably limit both oxygen uptake and carbon dioxide excretion. But one might also hypothesise that under these conditions the fish might compensate. A reduction of 15, 30 and 40% of total anatomical gill surface area in rainbow trout was accomplished by the ligation of 1, 2 and 3 gill arches, respectively. With the use of an extracorporeal shunt, arterial blood gases were continuously monitored during normoxia and graded hypoxia. As well, a dorsal aortic cannula allowed for periodic blood sampling for such variables as total arterial oxygen content (CaO2), total arterial carbon dioxide content (CaCO2), haemoglobin concentration, haematocrit and circulating catecholamines. Oxygen uptake (M˙O2), carbon dioxide excretion rates (M˙CO2) and ventilation convection requirements for both gases were determined through measurements of inspired and expired water carbon dioxide content (CCO2) and partial pressure of O2 (PO2) differences as well as in-flowing/out-flowing water CCO2, and PO2 differences. A final series of experiments examined the effects of carbonic anhydrase injections in ligated versus sham-ligated fish as well as comparing these results to ligated fish injected with physiological saline. (Abstract shortened by UMI.)

The main purpose of this thesis was to identify the changes that occur in m vim mammalian lipid metabolism during long-term fasting, arousal from hibernation, and following leptin treatment. Rabbits (Oryctolagus cuniculus) and woodchucks (Marmota monax) were fasted for one and two weeks, respectively, at room temperature and during different seasons. Woodchucks fasted in the summer lowered their energy expenditure and protein oxidation as soon as the fast was initiated. The woodchucks fasting in the spring did not, as their metabolic rate and protein utilization were already low, probably because in the wild they fast for extended periods of time following their final arousal from hibernation in the spring. The rabbits also exhibited metabolic depression and protein sparing with fasting although they used more protein and less lipid than the woodchucks. Using the continuous infusion method rates of triacylglycerol (TAG) hydrolysis (lipolysis), fatty acid (FA) mobilization and FA reesterification were measured on woodchucks that had just aroused from three months of hibernation. The contribution of TAGIFA substrate cycling to their total energy expenditure was extremely high (17%) Also, thermogenesis by this substrate cycle decreased the longer an animal had been aroused from hibernation, suggesting that this substrate cycle may be an important source of beat during arousal. Due to the high TAGIFA cycling rates, their lipolytic rate was also extremely high. Using the same methods, temporal changes in the rates of FA reesterification, lipolysis and lipid mobilization were measured in fasting rabbits. Their rates of lipolysis and FA mobilization initially doubled however, in the fix was prolonged, those rates decreased to pre-fasting levels. The metabolic depression induced by fasting probably accounted for the changes as less substrate was required for beta-oxidation. Reduced TAG/FA substrate cycling with long-term fasting accounted for a small amount of this metabolic depression (3%). The effects of leptin on TAG/FA recycling, lipolysis and FA mobilization were also investigated. In vivo leptin treatment caused significant increases in lipolytic rates and FA mobilization, and increased the relative contribution of lipid to the rabbit's total energy expenditure. The cycling rate of the TAG/FA substrate cycle was stimulated by leptin, contributing to 15% of the leptin-induced increase in energy expenditure. This showed that substrate cycling activation is a potent mechanism by which leptin can increase, metabolic thermogenesis.

The entry of bicarbonate ions (HCO3-) into the red blood cell (RBC) via the Cl-/HCO3 - exchanger for dehydration to CO2 has long been regarded as the limiting step of CO2 excretion in fish. The first component of this thesis was to test the hypothesis that proton availability may limit the catalysed dehydration of HCO3- within the extracellular compartment. The results presented in this thesis suggest that H+ availability does not appear to limit plasma HCO3- dehydration in vivo. Furthermore, the enhancement of CO2 excretion by means of the acceleration of HCO3- dehydration within the plasma following the administration of carbonic anhydrase (CA) demonstrated in this thesis was the first in vivo evidence ratifying the hypothesis of the chloride shift as the limiting factor on CO2 excretion in teleost fish. Using the knowledge acquired from the previous chapter, I tested the hypothesis that the slow entry rate of HCO3- into the RBC may be responsible for the apparent diffusion limitations on CO2 excretion. The experiments examined the effect of blood transit time through the gills on gas transfer to test the hypothesis that the efficiency of CO2 excretion is sensitive to changes in blood flow owing to chemical equilibrium limitations, whereas the efficiency of O2 uptake is insensitive to changes in blood flow. The insensitivity of PaO 2 to changes in cardiac output in vivo reaffirms the theory that O2 uptake behaves as a perfusion limited system. However, the sensitivity of CO2 transfer efficiency to changes in cardiac output, coupled with the impact of (CA) availability on this sensitivity, argue strongly that the apparent diffusion limitations on CO2 excretion are, in fact, chemical equilibrium limitations.

In order to examine the effects of exercise intensity on the post exercise elevation in esophageal temperature (DeltaTes), 8 male subjects performed 15 minutes of treadmill running exercise at 3 different exercise intensities, subthreshold (70% VO2max), threshold (84% VO 2max, and suprathreshold (93% VO2max), followed by an upright 45 minute recovery period. In addition, the effects of exercise intensity on the internal temperature threshold for cutaneous vasodilation (Tdil ), as well as post exercise blood pressure, cutaneous blood flow (SkBF) and cardiovascular conductance (DeltaCVC), were examined. Significant differences (p < 0.05) in DeltaTes were found between the 3 exercise intensities during exercise, as well as between suprathreshold (0.91°C), and threshold (0.56°C)/subthreshold (0.44°C) intensities during the recovery period. The elevated DeltaTes remained significantly above pre-exercise values for the entire recovery period for all 3 exercise intensities. Exercise intensity also demonstrated an effect on Tdil during exercise where significant differences were found between the 3 exercise intensities (0.26°C, 0.47°C, 0.84°C for subthreshold, threshold, and suprathreshold intensities respectively). However, no significant correlation was found between Tdil, and post exercise DeltaTes. Finally, a state of hypotension with respect to pre-exercise was observed during recovery from suprathreshold exercise. This could explain the significantly higher post exercise elevation in DeltaTes from subthreshold and threshold exercise.