The principle objective of this investigation was to develop an understanding of the biochemical and physiological response of free-ranging adult polar bears (<i>Ursus maritimus</i>) to prolonged fasting. A body condition index was developed from two measures, total body mass and straight-line body length, and was used as a covariate in the analyses of all other data. Protein and amino acid catabolism and urea synthesis were significantly lower in fasting bears when compared to feeding bears, and in fat bears when compared to lean bears. The inference from these results is that the energy metabolism in both states (fasting and fat) is one in which lipid is the predominant fuel for energy and nitrogen is conserved. Nutritional state (feeding versus fasting) had no significant effect on the plasma concentrations of non-esterified fatty acid, glycerol, and ketone bodies, or on the plasma ratio of acyl-carnitine to free carnitine. Furthermore, acetoacetate concentration was below the level of detection (<196 [mu]mol/L) in all bears, and â-hydroxybutyrate concentration never exceeded 291 [mu]mol/L. These results suggest polar bears are able to regulate closely the synthesis, release, and use of lipid metabolites without significant alteration in their plasma concentrations. Fasting polar bears showed no evidence of essential fatty acid (EFA) deficiency; the proportions of the diet-derived EFA linoleic (18:2[omega]6) and á-linolenic (18:3[omega]3) acids in the plasma and adipose tissue of fasting polar bears were greater than that in feeding polar bears. Plasma triiodothyronine concentrations and rectal temperatures were lower in fasting bears captured during summer-fall than in feeding bears, which suggests metabolic rates were decreased during fasting to conserve body fuels. Liver glycogen concentrations were found to be higher in fasting polar bears than in feeding bears. Furthermore, the results from intravenous administration of glucose (glucose tolerance test) to polar bears indicated the rates of insulin secretion and clearance in polar bears were slow relative to rates reported for other mammals. The inference from these results is that polar bears are not as dependent on glucose for energy as are other mammals and, as a consequence, are more lax in regulating their body glucose stores.

Hepatitis C virus (HCV) infects millions of people worldwide and is one of the leading causes of liver damage. Infection with HCV is strongly correlated with an increased risk of steatosis, or fatty liver disease, which is caused by a build-up of fat deposits in hepatocytes. All genotypes of HCV appear to cause some degree of steatosis in approximately 50% of infected individuals, especially in the presence of contributing host factors such as diabetes, obesity and alcoholism. However, approximately 70% of genotype 3a infections exhibit steatosis. Furthermore, successful clearance of the genotype 3a virus results in eradication of the steatosis, suggesting the genotype 3a virus may be able to directly cause steatosis.<p> Research suggests a role for the core protein of HCV, which forms the capsid of the virus, in the alteration of lipid metabolism pathways during infection. As such, I hypothesized that: 1) HCV alters lipid metabolism pathways and causes the build up of lipid in hepatocytes and the development of steatosis; 2) HCV-3a core protein has a differential or increased effect on these pathways in comparison to 1b core protein; and 3) other HCV proteins could also play a role in the altering of lipid metabolism. My research characterized the subcellular localization on lipid droplets of the HCV-3a core protein in comparison to HCV-1b core protein. It was found that HCV-3a core causes increased transcriptional activity from the Fatty Acid Synthase (FAS) promoter, an important enzyme involved in the synthesis of triglycerides in hepatocytes. In addition, one specific amino acid of HCV-3a core was determined to be partially responsible for this effect. Further research determined that the effect of HCV-3a core on FAS was dependent on the transcription factor Sterol Response Element Binding Protein-1 (SREBP-1) and the presence of HCV-3a core increased the processing and activity of SREBP-1. HCV core was also able to increase activity of Akt 1 and Akt2; inhibition of Akt activity resulted in decreased SREBP-1 activity thereby indicating that HCV core partially mediates SREBP-1 via Akt. Further experiments examined the role of another HCV protein, NS2, in these same lipid metabolism pathways. NS2 was also able to increase transcription from the FAS promoter via SREBP-1, suggesting that this HCV protein may also be important in the development of steatosis during HCV infection.<p> The evidence provided in these studies shows a very important role for HCV in altering lipid metabolism during infection that may lead to the development of steatosis. Current research suggests that the SREBP-1 pathway may be critical in the life cycle of the virus and these studies have provided important information on how lipid metabolism pathways are being changed by the virus. Hopefully this work can help identify potential treatment options for HCV that can slow down disease progression by preventing the development of steatosis or by decreasing viral replication.

We hypothesized that supplementation of dietary conjugated linoleic acid (CLA) and arginine singly or in combination would increase animal performance and meat quality by decreasing adiposity and increasing lean mass in growing-finishing pigs. Sixteen pigs (80 kg) were assigned to four treatments in a 2 x 2 factorial design, differing in dietary fatty acid and amino acid composition [control: 2.05% alanine (isonitrogenous control) plus 1% canola oil (lipid control); CLA: 2.05% alanine + 1% CLA; arginine: 1% arginine + 1% canola oil; arginine + CLA: 1% arginine + 1 CLA]. Preliminary tests indicated that up to 2% arginine was acceptable without interfering with lysine absorption. Pigs were allowed to feed free choice until reaching 110 kg. There were no significant differences across treatments in feed intake, weight gain, or feed efficiency. CLA tended to decrease carcass length (P = 0.06), whereas backfat thickness tended to be greater in pigs supplemented with arginine (P = 0.08). Arginine decreased muscle pH at 45 min postmortem (P = 0.001) and tended to increase lightness of muscle at 24 h postmortem (P = 0.07). CLA supplementation increased the concentrations of trans-isomers of 18:1 (P = 0.001) and SFA (P = 0.01) in s.c. and r.p. adipose tissue. CLA supplementation increased palmitate incorporation into total lipids in longissimus muscle (P = 0.01). Glucose oxidation to CO₂ in r.p. and s.c. adipose tissue were greater in pigs supplemented with CLA in the absence or presence of arginine (P = 0.03 and P = 0.04, respectively). The volume of s.c. adipocytes in s.c. and r.p. adipose tissues was greater in pigs supplemented with CLA, arginine, or CLA plus arginine than in control pigs (P = 0.001). Neither CLA nor arginine affected the expression of PGC-1[alpha],AMPK, mTOR, CPT-1A, FAS, or SCD (P > 0.05) in any tissues. We conclude that there was no significant interaction between arginine and CLA. Supplementary CLA or arginine to finishing-growing pigs did not modulate growth performance and did not reduce adiposity. CLA increased intramuscular fat content without deteriorating meat quality traits and increased saturated fatty acids and substrate oxidation in adipose tissues. In the presence of 1% of canola oil or CLA in the diet, arginine has the potential to deteriorate meat quality by reducing early postmortem pH and by increasing carcass fatness.

The aim of this study was to investigate the impact of Biomin P.E.P. – 1000 on the total quantity of lipids and triglycerides in blood serum of broiler chickens, also to analyze the impact of the preparation on the dynamics of growth and meat qaulity of birds. The results of our study show taht under the influence of phytogenic preparation Biomin P.E.P. – 1000 the content of total lipids and trigycerides in the blood serum of the broiler chickens at the age of 21 days increased, respectively, by 1,93 – 4,73 g/l and 0,22 – 1,07 mmol/l as compared to the control group. At the age of 42 days it increased: lipids by 0,82 – 1,61 g/l and triglycerides by 0,3 – 0,83 mmol/l as compared to the control group. In the influensce of this preparation the weight of broiler chickens increased by 9,92 – 10,62 % as compared to the control group. The testing of meat chemical composition showed positive influence of Biomin P.E.P. – on the quality of broiler chicken meat.

Suicide continues to claim hundreds of thousands of lives worldwide each year, in spite of the significant progress of research efforts aimed at understanding the complexity of this tragic behaviour. Data accumulated over the last decades suggest a certain biological predisposition to suicidal behaviour. Among the possible biological risk factors, cholesterol has frequently been cited. Several lines of evidence support the relationship between altered lipid metabolism, particularly low levels of serum cholesterol, and suicidal behaviour, yet the possible mechanisms governing the relationship remain to be elucidated. Three separate strategies were employed in order to explore the link between lipid metabolism and suicidal behaviour, each one from a novel perspective on this issue. The first approach aimed to substantiate the existing evidence of an association between low serum cholesterol and suicidality by examining psychiatric data, suicidality and related behavioural characteristics in a sample of Smith-Lemli-Opitz syndrome heterozygotes---a clinically normal population with altered cholesterol metabolism due to an inherited partial deficiency in the 7-dehydrocholesterol reductase enzyme---compared with controls. The second approach consisted in measuring the lipid profile in brain tissue from suicide completers, in order to address whether there are alterations in cholesterol and/or fatty acids in the brain. The final approach involved the use of exploratory gene expression studies to identify novel candidate genes and proteins that may be involved in mediating the link between lipid metabolism and suicidality. The results of these studies will be presented and discussed.

It is now well accepted, through the use of mutational studies, that genes influence behavioural variation. However, we have little knowledge of the cellular and neuronal mechanisms underlying the effects of specific genes. This thesis broadens our understanding of the neurogenetic underpinnings of naturally occurring differences in behaviour using the genetically tractable model organism Drosophila melanogaster. The thesis focuses on allelic variation at the foraging (for) gene which influences both larval and adult behaviour. In particular, for’s cellular/neural contributions to food-related behaviours and learning and memory is investigated. In the first study, we map FOR protein distribution patterns in the adult brain and use this knowledge to demonstrate a neural-specific function for the for gene in adult food-related behaviour. In the second study we demonstrate a novel role for for in the regulation of naturally existing differences in fly learning and memory in the mushroom bodies of the fly brain. In the third study, I explore FOR distribution patterns in larval tissues. I show that FOR is expressed both in neural and non-neural tissues suggesting a distributed function for FOR in food-related behaviours in the larva. In the last study, I describe naturally existing differences in fat metabolism in the Drosophila larva fat storage tissue. FOR is expressed in the fat storage tissue and may regulate lipid packaging, a trait linked to foraging. In general, my thesis is a cellular and neurogenetic analysis of natural variation in behavioural and physiological traits of D. melanogaster. The functions of FOR in food-related behaviours, nutrient physiology and cognition are conserved across taxa. The findings of this thesis should provide a framework to understand these phenomena in a wide range of organisms.

<p>Lipopolysaccharide, the physical barrier that protects Gram-negative bacteria from various antibiotics and environmental stressors, is anchored to the outer membrane by the phosphorylated, acylated disaccharide of glucosamine known as lipid A. Besides being necessary for the viability of most Gram-negative bacteria, lipid A interacts directly with specific mammalian immune cell receptors, causing an inflammatory response that can result in septic shock. The lipid A biosynthetic pathway contains nine enzymatic steps, the sixth being the phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) precursor to form lipid IV_A by the inner membrane-bound kinase LpxK, a divergent member of the P-loop containing nucleotide triphosphate hydrolase superfamily. LpxK is the only known P-loop kinase to act on a lipid at the membrane interface.</p><p> We report herein multiple crystal structures of <italic>Aquifex aeolicus</italic> LpxK in apo as well as ATP, ADP/Mg<super>2+</super>, AMP-PCP, and chloride-bound forms. LpxK consists of two &alpha;/&beta;/&alpha; sandwich domains connected by a two-stranded &beta;-sheet linker. The N-terminal domain, which has most structural homology to other P-loop kinase family members, is responsible for catalysis at the P-loop and positioning of the DSMP substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate using a 25º hinge motion about its base which also assembles the necessary catalytic residues at the active site.</p><p> Using a thin-layer chromatography-based radioassay, we have performed extensive kinetic characterization of the enzyme and demonstrate that LpxK activity <italic>in vitro</italic> is dependent on the presence of detergent micelles, the use of divalent cations, and formation of a ternary LpxK-ATP/Mg<super>2+</super>-DSMP complex. Implementing steady-state kinetic analysis of multiple point mutants, we identify crucial active site residues. We propose that the interaction of D99 with H261 acts to increase the pK_a of the imidazole group, which in turn serves as the catalytic base to deprotonate the 4&rsquo;-hydroxyl of DSMP. An analogous mechanism has not yet been reported for any member of the P-loop kinase family.</p><p> The membrane/lipid binding characteristics of LpxK have also been also investigated through a crystal structure of the LpxK-lipid IV_A product complex along with point mutagenesis of residues in the DSMP binding pocket. Critical contacts with the bound lipid include interactions along the glucosamine backbone and the 1-position phosphate group, especially through R171. Furthermore, analysis of truncation mutants of the N-terminal helix of LpxK demonstrates that this substructure is a critical hydrophobic contact point with the membrane, and that both charge-charge and hydrophobic interactions contribute to the localization of LpxK at the lipid bilayer. </p><p> Overall, this work has contributed significantly to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates. It has also provided insight into the process of enzyme evolution as LpxK, while containing a similar core domain as other P-loop kinases, has developed multiple subdomains required for both cellular localization and recognition of novel substrates. Finally, the presence of multiple crystal structures and detailed understanding of the LpxK catalytic mechanism will improve the chances of successfully targeting this essential step in lipid A biosynthesis in the pursuit of novel antimicrobials.</p> / Dissertation

It is now well accepted, through the use of mutational studies, that genes influence behavioural variation. However, we have little knowledge of the cellular and neuronal mechanisms underlying the effects of specific genes. This thesis broadens our understanding of the neurogenetic underpinnings of naturally occurring differences in behaviour using the genetically tractable model organism Drosophila melanogaster. The thesis focuses on allelic variation at the foraging (for) gene which influences both larval and adult behaviour. In particular, for’s cellular/neural contributions to food-related behaviours and learning and memory is investigated. In the first study, we map FOR protein distribution patterns in the adult brain and use this knowledge to demonstrate a neural-specific function for the for gene in adult food-related behaviour. In the second study we demonstrate a novel role for for in the regulation of naturally existing differences in fly learning and memory in the mushroom bodies of the fly brain. In the third study, I explore FOR distribution patterns in larval tissues. I show that FOR is expressed both in neural and non-neural tissues suggesting a distributed function for FOR in food-related behaviours in the larva. In the last study, I describe naturally existing differences in fat metabolism in the Drosophila larva fat storage tissue. FOR is expressed in the fat storage tissue and may regulate lipid packaging, a trait linked to foraging. In general, my thesis is a cellular and neurogenetic analysis of natural variation in behavioural and physiological traits of D. melanogaster. The functions of FOR in food-related behaviours, nutrient physiology and cognition are conserved across taxa. The findings of this thesis should provide a framework to understand these phenomena in a wide range of organisms.

The purpose of this study was to investigate whether a cyclic feeding regime alters growth, lipid content and the expression of genes involved in appetite regulation and lipid metabolism in rainbow trout (Onchorynchus mykiss). Progeny from two lots of diallel crosses were raised under a daily fed control or a cyclic feeding regime containing alternating reduced and compensatory rations. Although the cyclic feeding regime did not improve overall growth, there was some degree of compensatory growth seen during re-alimentation, and the reduced ration led to sustained reductions in condition factor and muscle fat content. Appetite-regulating genes showed little or no differences in expression between feeding regimes. However, the lipid metabolism genes involved in fatty acid synthesis and muscle lipid uptake showed differences in expression during reduced and compensatory ration. This study helps to clarify optimal cyclic feeding regimes in order to enhance growth characteristics preferential to the fish culture industry. / NSERC Strategic Grant, OMAFRA

Adipose tissue represents an important endocrine organ that secretes a multitude of adipokines known to mediate inflammation, lipid metabolism, and insulin sensitivity in peripheral tissues such as skeletal muscle. Specifically, adiponectin stimulates skeletal muscle fatty acid oxidation and is associated with improvements in insulin response. Long-chain n-3 polyunsaturated fatty acids (PUFA) are well known for their anti-inflammatory and insulin-sensitizing properties, and their dietary consumption is associated with a more favourable circulating adipokine profile, including increased adiponectin. However, whether n-3 PUFA can directly stimulate adiponectin secretion from human adipocytes, as well as the underlying mechanisms involved, is unknown. In contrast to n-3 PUFA, diets high in saturated fatty acids (SFA) are thought to decrease adiponectin and increase pro-inflammatory adipokines, as well as blunt skeletal muscle response to both adiponectin and insulin, possibly via activation of inflammatory pathways. The role of n-3 PUFA in mediating the communication between adipose tissue and skeletal muscle, as well as preventing SFA-induced impairments in skeletal muscle function, has yet to be examined. In this thesis, it was found that long-chain n-3 PUFA increase adiponectin secretion from human adipocytes via a peroxisome proliferator-activated receptor gamma-dependent mechanism. The effects of n-3 PUFA on adiponectin secretion were additive when combined with the thiazolidinedione, rosiglitazone. Secondly, incorporation of n-3 PUFA into a high SFA diet prevented impairments in adiponectin response and both prevented and restored impairments in insulin response in rodent skeletal muscle. Interestingly, these findings were paralleled by prevention of SFA-induced increases in toll-like receptor 4 expression by n-3 PUFA, suggesting inflammatory changes may be involved. Finally, dietary n-3 PUFA and SFA modulated the secretion of adipose tissue-derived factors from visceral rodent adipose tissue and subsequent exposure of isolated skeletal muscle to such factors induced acute changes in inflammatory gene expression without affecting insulin sensitivity. Together, the findings in this thesis suggest that n-3 PUFA modulate adipokine secretion from adipose tissue and that adipose-derived factors mediate skeletal muscle inflammation and response to adiponectin and insulin. Ultimately, this work highlights the importance of considering n-3 PUFA as a therapeutic strategy in the prevention and treatment of obesity and related pathologies.