During exposure to hypoxia, many vertebrates undergo behavioural, biochemical, and physiological changes in an attempt to overcome the lack of oxygen and thus maintain a constant metabolic rate. Numerous genes are known to be influenced by hypoxia in mammalian systems. Many of the genes that have been identified are similar to those found in fish. Two such genes are adrenomedullin and neuroglobin. Adrenomedullin is a multifunctional peptide that has shown to be expressed in a number of tissues. Neuroglobin is a recently discovered member of the globin family that is predominantly found in neuronal tissues and has a high affinity for oxygen. While the effects of hypoxia on these two genes are relatively well known in mammalian systems, they have not yet been examined in fish. To characterize the expression of both neuroglobin and adrenomedullin in different tissues and measure expressional changes during hypoxic exposure, adult zebrafish (Danio rerio) were exposed to hypoxic conditions (pO2 ∼23 torr or 3.07 kPa) for up to 24 hours. A variety of techniques were used, including in situ hybridization, immunohistochemistry, and real time PCR. Adrenomedullin was found to be present in all tissues examined except blood, the highest of which was found in the kidney and eye. Expression of neuroglobin was highest in the brain and eye; however, low levels of expression were also found in the gill, stomach and kidney. No significant changes in expression of either gene were observed following hypoxic exposure. In the brain, eye and gill, in situ hybridization and immunohistochemistry techniques were used to characterize expression of both genes at the protein and mRNA levels. Adrenomedullin mRNA staining was observed in the inner plexiform layer, outer nuclear layer and pigment epithelium of the eye, the chloride cells on the lamellae and in the interlamellar regions of the gill, and throughout all regions of the brain. Immunostaining revealed the presence of adrenomedullin protein in inner plexiform layer, outer plexiform layer and the pigment epithelium of the eye, apical membrane of lamellar epithelial cells of the gills and throughout all regions of the brain. Neuroglobin mRNA staining was observed in the ganglion cell layer as and the outer and inner nuclear layers of the eye, the chloride cells of the gill, and throughout all regions of the brain. Immunostaining revealed the presence of neuroglobin protein in the ganglion cell layer, pigment epithelium and photoreceptor cells as well as in the inner and outer plexiform layers of the eye, specific cells found throughout the gill filament, and in large neuronal cells as well as surrounding blood vessels of the brain.

This study tests the hypotheses that compensation for metabolic acid-base disturbances involves regulation of carbonic anhydrase (CA) and that cortisol plays an important role in mediating the compensatory responses of the rainbow trout, Oncorhynchus mykiss. Metabolic acidosis decreased mRNA expression of branchial trout cytosolic CA (tCAc) and increased mRNA expression of renal tCAc and membrane-bound CAIV (tCAIV). Metabolic alkalosis increased mRNA expression of branchial and renal tCAc, decreased renal tCAIV mRNA expression, and increased branchial CA activity. Plasma cortisol was elevated during acidosis and alkalosis, indicating its possible involvement in regulating the response to an acid-base challenge. Cortisol may up-regulate CA, as treatment with exogenous cortisol increased tCAc and tCAIV mRNA expression, renal tCAc protein abundance, and branchial CA activity. This study suggests that CA contributes to the compensation of metabolic acid-base disturbances in the rainbow trout, and that cortisol is involved in the regulation of acid-base status.

Exposure of an organism to a stressor results in a primary stress response. A part of this response involves the release of the catecholamines (CA), adrenaline (ADR) and noradrenaline (NADR), which exert their actions by binding to specific membrane binding sites or receptors termed adrenergic receptors or adrenoceptors (ARs). Continuous exposure to a hormone results in a decrease in the responsiveness of the cell. This process, termed desensitization may reduce the cell's responsiveness only to that hormone (homologous) or to another agonist (heterologous). Moreover, prolonged agonist exposure may result in the internalization (sequestration) and possible degradation of the receptor (down-regulation). These processes have been well studied for the three beta-AR subtypes in mammals, however little is known about how beta-ARs are regulated in teleost fish. The goal of this thesis was to examine the regulation of the beta-ARs in two species of teleosts, the rainbow trout (Oncorhynchus mykiss ) and the black bullhead (Ameiurus melas). Fish were exposed to various stressors including prolonged agonist exposure, physical exertion and hypercarbia and receptor binding characteristics [affinity (K d) and number of binding sites (Bmax)] as well as function were examined. Chasing to exhaustion and prolonged feeding of the beta-agonist clenbuterol significantly reduced the Bmax of hepatic beta 2-ARs in rainbow trout by 27% and 33%, respectively. In contrast, exposure to hypercarbia did not significantly affect rainbow trout gill beta-ARs. Pharmacological characterization of the gill beta-AR supports an atypical classification compared with the three classic mammalian subtypes. No significant differences were observed in binding parameters for bullhead hepatic beta-ARs after a 24 hour exposure to the beta-agonist isoproterenol or a 1 hour air-exposure. However, air-exposure did result in a moderate desensitization in CA-stimulated hepatic glucose production. Phylogenetic analysis groups the bullhead hepatic beta-AR with the mammalian beta3-ARs, which is not supported by the pharmacological classification of a beta2-AR subtype. This study was the first to comparatively examine 3-AR regulation for different subtypes of teleost beta-ARs. Findings suggest beta-ARs in teleosts possess different sensitivities to down-regulation and desensitization as is seen with the different mammalian beta-AR subtypes.

During environmental cold exposure in adult humans, a decrease in core temperature can be prevented by increasing heat production (H˙ prod) via shivering thermogenesis. The main purpose of this thesis was to determine the effects of changes in carbohydrate (CHO) availability and shivering intensity on oxidative fuel selection and muscle recruitment during cold exposure. Using a combination of metabolic and electrophysiological approaches, fuel selection and EMG activity were quantified: (i) during low-intensity shivering (LOW) in individuals with normal (N), low (LO) and high CHO availability (HI), and (ii) during high-intensity shivering in individuals with normal CHO availability (HIGH). Low-intensity shivering (2.6-fold increase in H˙prod) resulted in a stimulation of plasma glucose (+138%), muscle glycogen (+109%) and lipids (+376%) oxidation rates for N (CHAPTER 2). Despite the observed increase in plasma glucose oxidation, this fuel only supplied 10% H˙ prod (or only 25% of all the glucose oxidized). Total heat production was therefore unequally shared between lipids (50%), muscle glycogen (30%), plasma glucose (10%)and proteins (10%). The same fuel selection measurements were then performed for LO and HI (CHAPTER 3). The size of CHO reserves had no effect on H˙prod, but had a major impact on fuel selection before and during shivering. In the cold, a complete shift was observed from lipid oxidation for LO (53%, 28% and 19% H˙prod for lipids, CHO and proteins, respectively) to CHO-based metabolism for HI (23%, 65% and 12% H˙prod for lipids, CHO and proteins, respectively). As for N, plasma glucose oxidation was a minor fuel source (<13% H˙ prod), falling to 7% H˙prod for LO. Therefore, plasma glucose oxidation did not compensate for changes in muscle glycogen oxidation and thus is not a strategy used for maintaining heat production. Instead, proteins and lipids compensated for the decreased in CHO availability. Most interestingly, these drastic changes in fuel metabolism were achieved without altering the electromyographic (EMG) signature of shivering muscles (CHAPTER 4). Results demonstrate that EMG shivering intensity, pattern and spectral components of eight large muscles remains unaffected by changes in fuel selection. Therefore, humans can sustain low thermogenic rates by oxidizing widely different fuel mixtures within the same muscle fibers. Considering the low metabolic rates reached during mild shivering (LOW), oxidative fuel utilization rates were then measured at higher shivering intensity (3.5-fold rise in H˙prod) to establish whether (i) the role of plasma glucose could be increased (CHAPTER 5) (ii) modifications of fuel selection could be achieved via the recruitment of fuel specific muscle fibers. (Abstract shortened by UMI.)

Oncorhynchus mykiss epithelial calcium channel (ECaC) is thought to regulate calcium uptake across the gill. ECaC is a member of the transient receptor potential (TRP) gene family. In mammals, ECaC is divided into two sub-families, TRPV5 and TRPV6. Rainbow trout appears to possess only a single ECaC gene similar to the mammalian TRPV5 and TRPV6. Phylogenetic analysis suggests that an ancestral form of the gene diverged from those of lower vertebrates prior to a gene duplication event that gave rise to TRPV5 and TRPV6. In contrast to previous models of branchial calcium uptake which proposed an almost exclusive distribution of ECaC to mitochondrial rich cells (MRCs), the results of real time PCR on enriched gill cell populations as well as immunocytochemistry and in-situ hybridization analysis of enriched cells, cell cultures and whole gill sections suggest that ECaC is localized to a subset of both MRCs and pavement cells (PVCs). Using real time PCR and immunocytochemistry, the response of ECaC expression to various treatments known to increase or decrease Ca2+ uptake was examined. A significant increase in ECaC mRNA expression was observed following 5 days exposure of rainbow trout to softwater ([Ca2+] = 20--30 nmol 1-1). A concurrent increase in protein expression was suggested by the immunocytochemistry results (particularly in cells located at the tips of the lamellae) and confirmed by western blotting (identifying a single immunoreactive band at 90 kDa). A significant increase (almost 100-fold) in mRNA expression was also observed following exposure to 48 h of hypercapnia (∼7.5 mm Hg; a treatment known to increase Ca 2+ uptake capacity). Immunocytochemical analysis of the hypercapnic gills suggested an increase in the apical ECaC proteins on PVCs as well as on a subset of MRCs. Treatment of fish with cortisol caused a significant increase in ECaC expression. In contrast to the above experiments, induced hypercalcemia (through infusion of CaCl2, treatment known to reduce Ca2+ uptake) resulted in a significant reduction in ECaC gene expression. Taken together, the results presented in this thesis demonstrate that the levels of ECaC mRNA vary in direct relation to the Ca2+ transporting capacity of the gill (changes in transport capacity have previously been reported by other authors). These results thus provide evidence that, in part, Ca2+ uptake rates are regulated by the number of apical membrane Ca2+ channels that in turn modulate the inward flux of Ca2+ into gill epithelial cells. However, the data also suggests that a more in-depth characterization of gill cells is required since the distribution of ECaC appears to be more extensive than previously proposed.

Hypercalcaemia was induced artificially in American eels, Anguilla rostrata LeSueur, by infusing CaCl$\sb2.$ The treatment did not have any significant effect on recorded blood pressure or heart rate. Considering these data I concluded that, at least in vivo, the eel heart is apparently insensitive to physiological increases in plasma ultrafiltrable calcium concentration. The effects of changes in external calcium concentrations on the strength of contraction of isolated electrically-paced heart strips were also measured in rats, frogs, rainbowtrout and American eels. This was done to test the hypothesis that eel cardiac tissue demonstrates a relatively reduced sensitivity to changes in extracellular calcium concentration. The data obtained, however, showed that contrary to the hypothesis, the eel cardiac strips were generally more sensitive than those from the other animals tested. As a result the null hypothesis that eel hearts do not have reduced sensitivity to changes in extracellular calcium concentrations in vitro was accepted. In fact, on the basis of the in vitro data, one should conclude that eel cardiac tissue is very sensitive to extracellular calcium concentrations. This conclusion is confounded by the observation that the uptake rate of $\sp{45}$Ca measured in contracting and non-contracting eel heart muscle strips did not differ, an observation that suggests that the eel heart relies more on internal than on external calcium stores for the development of tension. We are thus left with an indeterminate situation. That is, whereas eel cardiac tissue is very sensitive to changes in extracellular calcium concentrations in vitro, in vivo the heart is able to mitigate the effects of alterations in extracellular (plasma) calcium concentrations. Future studies should focus on how this mitigation. or at least the attenuation, of the effect of extracellular calcium on cardiac function in vivo is obtained.

The objective of this work was to examine the influence of brown adipose tissue (BAT) on energy expenditure and body composition. Insights were gained from rodent models with BAT disruption, which is expected to contribute to obesity due to a deficit in energy expenditure for thermogenesis (Himms-Hagen 1989b). The capsaicin-desensitized (Cap-Des) rat has atrophied BAT with impaired thermogenesis, contradictorily leading to a decrease in body fat. Indeed, aging-associated obesity and hyperplasia of retroperitoneal white adipose tissue was prevented in 13.5 month old Cap-Des rats. The loss of function of Cap-sensitive afferent autonomic nerves (destroyed in Cap-Des rats) results in an alteration in energy balance conducive to leanness. It was suggested that the attenuated aging-associated increase in circulating calcitonin gene-related peptide (derived mainly from Cap-sensitive nerves) in the Cap-Des rat results in a lower degree of aging-associated insulin-resistance, hence in a lesser degree of obesity. Rats with a lesion in the nucleus of the solitary tract (NTS) are known to have an impaired capacity to increase their oxygen uptake and the temperature of their interscapular (I) BAT after administration of catecholamines. A similar impairment was seen in the Cap-Des rat, known also to sustain a lesion in the NTS, and associated with marked atrophy of IBAT. The disruption of the NTS did not cause BAT atrophy by the parameters measured; atrophy of BAT in Cap-Des rats is not due to the NTS-lesion that they sustain. UCP-DTA transgenic mice have a partial ablation of BAT, accomplished by linking DTA (diphtheria toxin A chain) to the uncoupling protein1 (UCP1) promoter. UCP1-expressing brown adipocytes are destroyed in the UCP-DTA transgenic mouse when the tissue is stimulated. As expected, these mice develop massive obesity; also present are hyperphagia, insulin resistance, hyperglycemia, hyperinsulinemia, and hyperlipidemia characteristic of noninsulin-dependent diabetes mellitus (Lowell et al. 1993). UCP-DTA and control mice were raised at thermoneutrality (35$\sp\circ$C) in order to suppress BAT thermogenesis. As predicted, eliminating the deficit in BAT thermogenesis present in the UCP-DTA mice corrected their obesity and hyperphagia. UCP is not totally eliminated in the UCP-DTA mice, as measured by an antibody that detects UCP1, UCP2 (and probably UCP3), and these mice are not sensitive to cold (Lowell et al. 1993). Mice were acclimated to mild cold (14$\sp\circ$C) to determine the nature of compensation for the lack of UCP1-expressing brown adipocytes in UCP-DTA mice. BAT does grow in cold-adapted UCP-DTA mice, perhaps by substitution of brown adipocytes expressing a different UCP isoform for brown adipocytes that would normally express UCP1. Adjustment of food intake in accordance with environmental temperature was shown to occur independent of changes in serum leptin concentration. Intact BAT was shown to be necessary for the control of food intake at temperatures below thermoneutrality. Because the lack of UCP1-mediated thermogenesis in BAT of knockout mice is known not to induce hyperphagia, we propose the deficiency of UCP1-expressing brown adipocytes, specifically the absence of a postulated satiety factor normally secreted from them, is responsible for the hyperphagia of the UCP-DTA mice at 24$\sp\circ$C.

The present study investigated the paracrine/autocrine role of prostaglandins in the development of the adipocyte phenotype. To determine the paracrine role of adipocyte-derived prostaglandins on differentiation of preadipocytes, test preadipocytes were cultured with medium conditioned by isolated mature adipocytes (ACM). ACM has been shown by our laboratory to contain factors released by mature adipocytes which induce differentiation. Addition of indomethacin, an inhibitor of prostaglandin synthesis, to the culture of adipocytes (ACM+I) significantly augmented differentiation induced by ACM. (p $\le$ 0.05). In contrast, addition of indomethacin to preadipocytes (ACM+Ia) resulted in significant inhibition of differentiation (p $\le$ 0.05) induced by ACM and standard differentiation mixture (MIX). These findings suggested that adipocytes secrete prostaglandins that may act as negative paracrine regulators of preadipocyte differentiation, whereas, preadipocytes may secrete prostaglandins that stimulate their differentiation in an autocrine manner. Cultured adipocytes and preadipocytes released PGI$\sb2$ and PGE$\sb2$ into medium. However, the amount of PGI$\sb2$ and PGE$\sb2$ released by preadipocytes was 1.4- and 11-fold greater than those released by adipocytes. When the effect of exogenous prostaglandins on preadipocyte differentiation was examined, PGI$\sb2$ and PGE$\sb2$ stimulated preadipocyte differentiation, but the effective concentration $({\ge}10\sp{-7}$ M) was much higher than the concentration found to be released in the culture medium of adipocytes. PGF$\sb{2\alpha},$ in contrast, inhibited differentiation significantly (p $\le$ 0.05) at a lower concentration $(10\sp{-8}$ M). These findings suggested that adipocytes may release PGE$\sb2$ and PGI$\sb2$ in amounts that are insufficient to stimulate preadipocyte differentiation. Hence the differentiating promoting activity of ACM cannot be due to the presence of PGI$\sb2$ or PGE$\sb2.$ (Abstract shortened by UMI.)

The overall objective of this thesis was to examine various aspects of the metabolic significance and regulation of the mitochondrial proton leak. The research conducted specifically assesses the influence that leak has on age-associated changes in mitochondria, and the role that the leak plays in facultative energy expenditure of transgenic mice which lack uncoupling protein-1 (UCP1), a well known mediator of the proton leak. Proton leak in mitochondria has been studied for over ten years, but its exact mechanism has not yet been elucidated and only recently has it been realized that it might be mediated by uncoupling proteins (UCPs). UCPs may confer a mechanism for proton leakage and thus affect the efficiency of oxidative phosphorylation. Mice deficient in the gene for mitochondrial UCP1 (Ucp1-deficient mice) are cold-sensitive despite their abundant expression of genes for the isoforms (Ucp2 and Ucp3), and do not become more obese than controls when fed a high fat diet (Enerback et al. 1997) The objective of our work was to analyse the metabolic control and characteristics of proton leak in mitochondria from brown adipose tissue (BAT) of Ucp1-deficient mice and of heterozygote controls in order to establish the role of the UCPs in facultative thermogenesis. (Abstract shortened by UMI.)

Proliferation and differentiation of tubular cells occur after renal ischemia/reperfusion. Angiotensin II has multiple growth effects on different renal cell types, and also regulates renal blood flow In particular, proximal tubular cells locally synthesize angiotensin II, which can bind to AT$\sb1$ receptors in an autocrine/paracrine fashion. The function of the AT$\sb2$ receptor is unknown, but is linked to apoptosis in other tissues. In the current study, gene expression for intrarenal angiotensinogen, cortical AT$\sb1$ receptor and proximal tubular AT$\sb1$ receptor was determined by Northern blots, and gene expression for the AT$\sb2$ receptor was determined by RT-PCR. In rats after ischemia/reperfusion. A significant downregulation of angiotensinogen mRNA occurred from 6 to 72 hours post-ischemia/reperfusion, whereas the cortical AT$\sb1$ receptor did not change significantly. In contrast, proximal tubular AT$\sb1$ receptor mRNA significantly decreased from 0 to 24 hours post-ischemia/reperfusion, and recovered after 72 hours. New gene expression for the AT$\sb2$ receptor was observed in ischemic proximal tubular segments and in the ischemic outer medulla 120 hours after ischemia/reperfusion. By histoautoradiography, a significant decrease in the density of angiotensin II binding was noted in the ischemic cortex after 24 hours post-ischemia/reperfusion, and in the outer medulla at 3 and 24 hours. At 120 hours post-ischemia/reperfusion, intrarenal angiotensin II levels, density of angiotensin II binding and angiotensinogen gene expression recovered. In conclusion, the intrarenal renin-angiotensin system is downregulated with ischemia/reperfusion, and recovers 120 hours post-ischemia/reperfusion, with co-expression of AT$\sb1$ and AT$\sb2$ receptor mRNA at this time. These findings suggest that co-expression of AT$\sb1$ and AT$\sb2$ receptors may be involved in the remodelling of injured tissue.