Knowledge on the precise effects of surplus glucose supply in dairy cows is limited by the lack of information on how intermediary metabolism adapts at different levels of glucose availability. Therefore, a gradual increase of glucose supply via intravenous glucose infusion was used in the present study to test the dose effect of surplus provision of glucose on the metabolic status and milk production of dairy cows. Furthermore, the effects of increasing levels of surplus glucose on mRNA expressions and activities of rate-limiting enzymes involved in hepatic gluconeogenesis were investigated. Based on a previous finding that a positive energy balance may decrease hepatic carnitine palmitoyltransferase (CPT) enzyme activity, it was also of interest whether skeletal muscle CPT activity is downregulated in a similar manner during positive energy balance. Twelve midlactating Holstein-Friesian dairy cows were continuously infused over a 28-d experimental period with either saline (SI group, six cows) or 40% glucose solutions (GI group, six cows). The infusion dose was calculated as a percentage of the daily energy (NEL) requirements by the animal, starting at 0% on d 0 and increasing gradually by 1.25%/d until a maximum dose of 30% was reached by d 24. Dose was then maintained at 30% NEL requirement for 5 d. No infusions were made between d 29-32. Liver and skeletal muscle biopsies were taken on d 0, 8, 16, 24, and 32. Body weight (BW) and back fat thickness (BFT) were recorded on biopsies days. Blood samples were taken every 2 d. In addition, blood samples over 24 h (6-h intervals) were taken the days before each biopsy. Milk and urine samples were taken on biopsies days. BW and BFT increased linearly with increasing glucose dose for GI cows. No differences were observed in the dry matter intake, milk energy output, and energy corrected milk yield between groups. However, milk protein percentage and yield increased linearly in the GI group. Only occasional increases in blood glucose and insulin concentrations were observed in blood samples taken at 1000 h every 2 d. However, during infusion dose of 30% NEL requirements on d 24, GI cows developed postprandial hyperglycemia associated with hyperinsulinemia, coinciding with glucosuria. The revised quantitative insulin sensitivity check index (RQUIKI) indicated linear development of insulin resistance for the GI treatment. GI decreased serum concentrations of beta-hydroxybutyrate (BHBA) and blood urea nitrogen and tended to decrease the serum concentration of non-esterified fatty acids (NEFA). Liver glycogen content increased, while glycogen content in skeletal muscle only tended to increase by GI. No significant changes were observed in the activities and relative mRNA expression levels of hepatic phosphoenolpyruvate carboxykinase and glucose 6-phospatase. The activity of fructose 1,6-bisphosphatase (FBPase) and relative mRNA expression levels of pyruvate carboxylase (PC) were decreased in the GI group but only during the high dose of glucose infusion. Hepatic CPT activity decreased with GI and remained decreased on d 32. The hepatic expression levels of CPT-1A and CPT-2 mRNA were not significantly altered but tended to reflect the changes in enzyme activity. No effect of glucose infusion was observed on skeletal muscle CPT activity. The aforementioned adaptations were reversed four days after the end of glucose infusions except for those of BW, BFT, and lipid metabolism (i.e. serum BHBA and NEFA concentrations, hepatic CPT activity). It is concluded that mid-lactation dairy cows on an energy-balanced diet direct intravenously infused glucose predominantly to body fat reserves but not to increased lactation performance. Cows rapidly adapted to increasing glucose supply but experienced dose-dependent development of insulin resistance corresponding with postprandial hyperglycemia/hyperinsulinemia and glucosuria at dosages equivalent to 30% NEL requirements. The catalytic capacity of key hepatic gluconeogenesis enzymes in mid-lactating dairy cows is not significantly affected by nutritionally relevant increases of glucose supply. Only very high dosages selectively suppress PC transcription and FBPase activity. Finally, it can be concluded that suppression of CPT activity by positive energy balance appears to be specific for the liver in midlactating dairy cows.

Background: Long QT syndrome (LQTS) is a cardiac condition which predisposes individuals to syncope, seizures, and sudden cardiac death. There is a high prevalence of congenital LQTS in a First Nations community in Northern British Columbia due to the founder variant p.V205M in the KCNQ1 gene. Additionally, two other variants of interest are present in this population: the KCNQ1 p.L353L variant, previously noted to modify the phenotype of LQTS in adults, and the CPT1A p.P479L variant, a metabolic variant common in Northern Indigenous populations associated with hypoglycemia and sudden unexpected infant death. Methods: We performed a mixed methods study to better understand the impact of LQTS in children and youth in this First Nations community. To learn about the clinical impact of LQTS, and better understand the effects of the KCNQ1 and CPT1A variants in children, we used statistical analysis to compare the cardiac phenotypes of 211 First Nations children with and without the p.V205M, p.L353L and p.P479L variants, alone and in combination. Ordinary Least Squares linear regression was used to compare the highest peak corrected QT interval (QTc). The peak QTc is an electrocardiogram measurement used in risk stratification of LQTS patients. Logistic regression was used to compare the rates of syncope and seizures experienced in childhood. Additionally, to learn about the lived-experience of LQTS, we interviewed one young First Nations adult about her experiences growing up with LQTS as a teenager. From this interview, we conducted a qualitative case study analysis using Interpretative Phenomenological Analysis. All research was done in partnership with the First Nations community using community-based participatory methods. Results: We found that the p.V205M variant conferred a 22.4ms increase in peak QTc (p<0.001). No other variants or variant interaction effects were observed to have a significant impact on peak QTc. No association between the p.V205M variant and loss of consciousness (LOC) events (syncope and seizures) was observed (OR(95%CI)=1.3(0.6-2.8); p=0.531). However, children homozygous for p.P479L were found to experience 3.3 times more LOC events compared to non-carriers (OR=3.3(1.3-8.3); p=0.011). With regard to the qualitative portion of the thesis, four superordinate (main) themes emerged from the case study: Daily life with Long QT Syndrome, Interactions with Medical Professionals, Finding Reassurance, and The In-Between Age. We found that even though our participant was asymptomatic and felt that she was not impacted by LQTS in her daily life, she considered certain elements of the condition to be stressful, such as taking a daily beta-blocker. Conclusion: These results suggest that while the KCNQ1 p.V205M variant is observed to significantly prolong the peak QTc, the CPT1A p.P479L variant is more strongly associated with LOC events in children from this community. More research is needed to further determine the effect of these variants; however, our preliminary findings suggest management strategies, such as whether beta-blockers are indicated for p.V205M carriers, may need to be reassessed. The importance of developing a holistic, well-balanced approach to medical care, taking into consideration the personal perspectives and unique medical circumstances of each child is exemplified in this study. / Graduate

Il est désormais accepté qu'un environnement foetal défavorable prédispose à des maladies chroniques qui surviennent à l'âge adulte. Il a été démontré dans notre laboratoire qu'une diminution de perfusion placentaire induit une redistribution du débit sanguin vers le coeur chez le foetus ainsi qu’une restriction de croissance intrautérine. De plus, un remodelage et une diminution de la contractilité des cardiomyocytes ont été observés chez les femelles devenues adultes. En période périnatale, l’utilisation des acides gras comme substrat énergétique devient plus importante que celle du glucose au niveau des cardiomyocytes. Considérant qu'un mécanisme s'est mis en place in utero, nous émettons l’hypothèse que le transfert de la voie de l’utilisation du glucose vers l’utilisation des acides gras se fait plus tôt chez les foetus en restriction de croissance. L’objectif de cette étude est de mesurer, dans les coeurs foetaux, les constituants du métabolisme des acides gras, soit le transporteur principal des acides gras, la carnitine palmitoyltransférase‒1‒alpha, ainsi que ses protéines associées soit l’acyl‒CoenzymeA synthétase‒1 et le canal anionique voltage‒dépendant de type 1. Nous mesurerons l’activité du cytochrome c oxydase et le nombre de mitochondries. L’influence du sexe et la condition foetale (restriction de croissance intrautérine vs contrôle) seront comparés. Nous avons observé que l’expression protéique de la carnitine palmitoytransférase‒1α et de l’acyl‒CoenzymeA synthétase‒1 est significativement augmentée, mais pas celle du canal anionique voltage‒dépendant de type 1, dans les coeurs de foetus en restriction de croissance intrautérine femelles. Le nombre et l’activité des mitochondries est semblable dans tous les groupes. Ces résultats suggèrent que la condition foetale et le sexe altèrent la quantité du transporteur des acides gras, la carnitine palmitoytransférase‒1α, au niveau traductionnel sans toutefois affecter l’activité du cytochrome c oxydase et le nombre de mitochondries. À long terme, nos études permettront de mieux comprendre les conséquences et causes de la RCIU afin d’en permettre la prévention. / It is widely accepted that an adverse fetal environment predisposes to chronic diseases that occur in adulthood. In our laboratory, it has been shown that a decrease in placental perfusion induced blood flow redistribution to the heart in fetus as well as an intrauterine growth restriction. Furthermore, remodeling and decreased contractility of cardiomyocytes were observed in adult offspring females. In perinatal period, the use of fatty acids as an energy substrate becomes more important than glucose in cardiomyocytes. Whereas a mechanism is in place in utero, we proposed the hypothesis that the transfer from the use of glucose as fuel towards fatty acids occurs earlier in intrauterine growth restriction fetuses. The objective of this study is to measure, in fetal hearts, the components of fatty acid metabolism, the main carrier of fatty acids, carnitine palmitoyltransférase‒1α, and its associated proteins namely acyl‒coenzymeA synthase‒1 and voltage‒dependant anion channel 1. We will measure the cytochrome c oxidase activity and the number of mitochondria. Influence of sex and fetal condition (intrauterine growth restriction vs control) will be compared. We observed that protein expression of carnitine palmitoyltranferase-1α and acyl-coenzyme A synthetase long-chain 1 is significantly increased, but not that of the voltage-dependant anion channel 1, in hearts of female fetus IUGR. The number and the activity of mitochondria are similar in all the groups. These results suggest that fetal condition and sex alter the quantity of the acid transporter carnitine palmitoyltranferase-1α has at the translation level without affecting the activity of the cytochrome c oxidase and the number of mitochondria.

Decline in cardiac pump function is a hallmark of aging where mitochondrial decay is an important underlying cause. Although certainly multifactorial in nature, both dysfunction of the machinery involved in the chemiosmotic process of energy transduction and lower capacity to maintain fatty acid-driven respiration are identified as intrinsic factors of mitochondrial decay in the aged myocardium. Age-associated destabilization of electron transport supercomplexes as a potential factor of mitochondrial decay in the rat heart. Defective operation of the electron transport chain (ETC) constitutes a key mechanism involved in the age-associated loss of mitochondrial energy metabolism. Nevertheless, the molecular events underlying inefficient electron flux that ultimately leads to higher superoxide appearance and impaired respiration are not fully known. As recent biophysical evidence shows that the ETC may form large macromolecular assemblies (i.e. supercomplexes) that disintegrate in certain pathologies (e.g. heart failure or Barth syndrome) reminiscent of aging, we investigated the hypothesis that alterations in supercomplexes are partly responsible for the age-related loss of cardiac ETC function. In this dissertation, age-associated changes in supercomplex organization and stability were investigated in subsarcolemmal (SSM) and interfibrillary (IFM) mitochondria isolated from cardiac tissue from young (3-5 months) and old (24-28 months) male Fischer 344 rats. Blue native-PAGE (BN-PAGE) analysis of digitonin-solubilized mitochondrial membranes coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to investigate supercomplex organization. Results show that both SSM and IFM display supercomplexes comprised of various stoichiometries of complexes I, III and IV (never complex II), which typically organize as high mass (1500-2300 kDa) assemblies containing up to four copies of complex IV (i.e. I���III���IV[subscript N]-type supercomplexes). Interestingly, analysis of IFM proteins showed that, in general, supercomplex levels declined by up to 15 % (p < 0.05) with age; however, different degrees of supercomplex deterioration were observed, depending on the particular supercomplex investigated. Supercomplexes of the highest molecular weights (i.e. 1900-2300 kDa), which were also composed of the most complex stoichiometries (i.e. I1III2IVN, N ��� 2), were primarily lost with age. In particular, I���III���IV���, I���III���IV��� and I���III���IV��� supercomplexes were found to decline by 13% (p < 0.05), 30% (p < 0.05) and 45% (p < 0.05), respectively, on an age basis. Therefore, the age-associated loss of supercomplexes in IFM stems from destabilization of the assemblies that comprise several copies of complex IV, which could partially limit proper electron transfer to O��� for its reduction, affecting mitochondrial respiratory capacity. In contrast to IFM, the aging defects of SSM supercomplexes appeared to be confined to the assembly comprised of only one copy of complex IV (I���III���IV���, 1700 kDa) (37% loss; p = 0.06), while the higher molecular weight supercomplex sub-types that were most affected in IFM (i.e. I���III���IV[subscript N], N ��� 2) were not significantly altered with age. Thus, the results from this dissertation indicate that mitochondria from different subcellular locations in the myocyte show different degrees of supercomplex destabilization in the aging rat heart. The more robust supercomplex deficits noted for IFM fit well with previous observations that electron transport characteristics of this subpopulation are more adversely affected with age than SSM. Although the underlying factor(s) of supercomplex deterioration are not fully known, the hypothesis that age-related alterations of certain constituents of the IMM (e.g. cardiolipin) may be important factors of supercomplex destabilization in cardiac mitochondria was investigated in this dissertation. To this end, LC-MS/MS characterization of supercomplex proteins and HPLC analysis of cardiolipin were used as approaches to elucidate potential factor(s) of supercomplex destabilization in the aging rat heart. Age-related alterations of cardiolipin levels and its acyl-chain content showed a strong parallel to the age-associated destabilization of supercomplexes. Specifically, cardiolipin levels declined by 10% (p < 0.05) in IFM, the mitochondrial subpopulation displaying the highest degree of supercomplex deterioration. In addition, the content of (18:2)���-cardiolipin, the predominant species in the heart, was found to decline by 50% (p < 0.05) on average in both populations of cardiac mitochondria. Therefore, the data presented in this dissertation indicate that changes in cardiolipin may be at least one of the factors involved in supercomplex destabilization in the aging heart. Age-related decline in carnitine palmitoyltransferase I (CPT1) activity as a mitochondrial lesion that limits fatty acid catabolism in the rat heart. Loss of fatty acid utilization, another intrinsic factor of mitochondrial decay in the aged myocardium, has been associated with age-related alterations in the activity of carnitine palmitoyltransferase 1 (CPT1), the rate-controlling enzyme for overall fatty acid ��-oxidation. Nevertheless, the exact molecular mechanism involved in the age-related loss of fatty acid-driven bioenergetics is not fully understood. In this dissertation, it was also investigated whether the aging lesion for fatty oxidation lies in a particular mitochondrial subpopulation or more generally results from cardiac decrements in L-carnitine levels. In order to clarify the role of each one of these factors, the effect of long-term dietary supplementation with the L-carnitine analogue, acetyl-L-carnitine (ALCAR), was also investigated. Results show that aging selectively decreases CPT1 activity in IFM by reducing enzyme catalytic efficiency for palmitoyl-CoA. IFM displayed a 28% (p < 0.05) loss of CPT1 activity, which correlated with a decline (41%, p < 0.05) in palmitoyl-CoA-driven state 3 respiration. Interestingly, SSM had preserved enzyme function and efficiently utilized palmitate. Analysis of IFM CPT1 kinetics showed both diminished V[subscript max] and K[subscript m] (60% and 49% respectively, p < 0.05) when palmitoyl-CoA was the substrate. However, no age-related changes in enzyme kinetics were evident with respect to L-carnitine. ALCAR supplementation restored CPT1 activity in heart IFM, but not apparently through remediation of L-carnitine levels. Rather, ALCAR influenced enzyme activity over time, potentially by modulating conditions in the aging heart that ultimately affect palmitoyl-CoA binding and CPT1 kinetics. In conclusion, this dissertation presents a characterization of age-associated alterations in the macromolecular organization of the IMM components that could partly explain the loss of mitochondrial oxidative capacity that affects the aging heart. In addition, the characterization of an age-related lesion of the controlling enzyme for ��-oxidation is presented as another important factor that limits mitochondrial function and energy metabolism in cardiac mitochondria. / Graduation date: 2013

Artificial divergent selection of chickens for high and low body weight at 8 weeks of age has produced two lines: the high (HWS) and low (LWS) body weight chicken lines. In addition to the difference in body weight, the lines show extreme differences in feeding behaviour and body composition. The aim of this study was to uncover the genetic and molecular factors that contribute to and determine these differences, especially regarding body energy regulation and appetite. In papers I and II, genome-wide gene expression in a brain sample containing hypothalamus and in dissected hypothalamus was analysed using DNA microarray and qRT-PCR. We found that levels of differential expression were generally moderate, which was consistent with the idea that polygenic factors were involved in the establishment of the chicken lines. Genes associated with neural plasticity, lipid metabolism and body energy regulation were differentially expressed. This result indicated that the neural systems regulating feeding behaviour and body weight were altered in the chicken lines. However, genes that were involved in the central melanocortin system were not systematically differentially expressed. Interestingly, the biggest differences in expression between the lines found in endogenous retrovirus sequences of the ALV subgroup E. Thus, in paper III, we characterized the number of integrations, the expression of ALVE retroviral elements and their effects on body weight. A significant correlation between low body weight and high ALVE expression was observed in female F9 birds from an HWS x LWS advanced intercross line. This implied that ev-loci contributing to increased ALVE expression levels were genetically linked to loci influencing the low body weight of the pullets. In paper IV, the carnitine palmitoyltransferase-1b gene (CPT1B), which was highly differentially expressed in the hypothalami, was investigated. We mapped chicken CPT1B to the distal tip of chromosome 1p. The levels of CPT1B mRNA in the HWS line were higher in the hypothalamus and lower in muscle than in the LWS line. This pattern of differential expression indicates that this gene could contribute to the remarkable phenotypic differences between HWS and LWS chickens. However, comparison with quantitative trait loci data showed that the expression of CPT1B is a trans effect, rather than a direct causative locus. In conclusion, the data suggested that the long-term selection for body weight resulted in differential gene expression in the brains of the selected chicken lines. These results may have relevance for the poultry industry and will also contribute to increasing knowledge about human diseases such as obesity and anorexia.

Knowledge on the precise effects of surplus glucose supply in dairy cows is limited by the lack of information on how intermediary metabolism adapts at different levels of glucose availability. Therefore, a gradual increase of glucose supply via intravenous glucose infusion was used in the present study to test the dose effect of surplus provision of glucose on the metabolic status and milk production of dairy cows. Furthermore, the effects of increasing levels of surplus glucose on mRNA expressions and activities of rate-limiting enzymes involved in hepatic gluconeogenesis were investigated. Based on a previous finding that a positive energy balance may decrease hepatic carnitine palmitoyltransferase (CPT) enzyme activity, it was also of interest whether skeletal muscle CPT activity is downregulated in a similar manner during positive energy balance. Twelve midlactating Holstein-Friesian dairy cows were continuously infused over a 28-d experimental period with either saline (SI group, six cows) or 40% glucose solutions (GI group, six cows). The infusion dose was calculated as a percentage of the daily energy (NEL) requirements by the animal, starting at 0% on d 0 and increasing gradually by 1.25%/d until a maximum dose of 30% was reached by d 24. Dose was then maintained at 30% NEL requirement for 5 d. No infusions were made between d 29-32. Liver and skeletal muscle biopsies were taken on d 0, 8, 16, 24, and 32. Body weight (BW) and back fat thickness (BFT) were recorded on biopsies days. Blood samples were taken every 2 d. In addition, blood samples over 24 h (6-h intervals) were taken the days before each biopsy. Milk and urine samples were taken on biopsies days. BW and BFT increased linearly with increasing glucose dose for GI cows. No differences were observed in the dry matter intake, milk energy output, and energy corrected milk yield between groups. However, milk protein percentage and yield increased linearly in the GI group. Only occasional increases in blood glucose and insulin concentrations were observed in blood samples taken at 1000 h every 2 d. However, during infusion dose of 30% NEL requirements on d 24, GI cows developed postprandial hyperglycemia associated with hyperinsulinemia, coinciding with glucosuria. The revised quantitative insulin sensitivity check index (RQUIKI) indicated linear development of insulin resistance for the GI treatment. GI decreased serum concentrations of beta-hydroxybutyrate (BHBA) and blood urea nitrogen and tended to decrease the serum concentration of non-esterified fatty acids (NEFA). Liver glycogen content increased, while glycogen content in skeletal muscle only tended to increase by GI. No significant changes were observed in the activities and relative mRNA expression levels of hepatic phosphoenolpyruvate carboxykinase and glucose 6-phospatase. The activity of fructose 1,6-bisphosphatase (FBPase) and relative mRNA expression levels of pyruvate carboxylase (PC) were decreased in the GI group but only during the high dose of glucose infusion. Hepatic CPT activity decreased with GI and remained decreased on d 32. The hepatic expression levels of CPT-1A and CPT-2 mRNA were not significantly altered but tended to reflect the changes in enzyme activity. No effect of glucose infusion was observed on skeletal muscle CPT activity. The aforementioned adaptations were reversed four days after the end of glucose infusions except for those of BW, BFT, and lipid metabolism (i.e. serum BHBA and NEFA concentrations, hepatic CPT activity). It is concluded that mid-lactation dairy cows on an energy-balanced diet direct intravenously infused glucose predominantly to body fat reserves but not to increased lactation performance. Cows rapidly adapted to increasing glucose supply but experienced dose-dependent development of insulin resistance corresponding with postprandial hyperglycemia/hyperinsulinemia and glucosuria at dosages equivalent to 30% NEL requirements. The catalytic capacity of key hepatic gluconeogenesis enzymes in mid-lactating dairy cows is not significantly affected by nutritionally relevant increases of glucose supply. Only very high dosages selectively suppress PC transcription and FBPase activity. Finally, it can be concluded that suppression of CPT activity by positive energy balance appears to be specific for the liver in midlactating dairy cows.