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A metabolic study was conducted with 23 men to determine the effects on plasma lipids and lipoproteins of a high calcium intake from two sources compared to a normal level of calcium intake over an eight week controlled feeding period. Three diet treatments were examined: 1) high dietary calcium mainly from dairy sources (1600-1800 mg/day), 2) high dietary calcium supplied by a CaCO₃ supplement (1600-1800 mg/day), and 3) normal dietary calcium intake from mixed sources (600-800 mg/day). Fat, carbohydrate and protein were provided in the ratio of percent kcal as 40:49:11. Nutrient, cholesterol levels (500 mg) and polyunsaturated to saturated (P/S) fatty acid ratio (0.446) were held constant for all diet treatments. There were no significant differences between treatments or across time in plasma TC, LDL, HDL or VLDL-cholesterol. Levels remained similar throughout the controlled diet treatment and the pre- and post-treatment periods. At the level of fat content and P/S ratio tested, there was no hypocholesterolemic effect of elevated calcium intake on plasma lipids. / Master of Science

Chiu Chi Pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 121-134). / Abstracts in English and Chinese. / DECLARATION --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABBREVIATIONS --- p.iii / ABSTRACT --- p.vi / 摘要 --- p.viii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Cholesterol --- p.1 / Chapter 1.1.1 --- History of cholesterol --- p.1 / Chapter 1.1.2 --- Structure of cholesterol --- p.1 / Chapter 1.1.3 --- Biological function of cholesterol --- p.3 / Chapter 1.1.4 --- Sources of cholesterol in our body --- p.3 / Chapter 1.2 --- Lipid hypothesis --- p.4 / Chapter 1.2.1 --- Relationship between dietary cholesterol and plasma cholesterol --- p.4 / Chapter 1.2.2 --- "Hypercholesterolemia , atherosclerosis and coronary heart disease (CHD)" --- p.4 / Chapter 1.2.3 --- Individual variation --- p.5 / Chapter 1.3 --- Cholesterol homeostasis --- p.7 / Chapter 1.3.1 --- SREBPs up-regulates the expression of LDL-receptor and HMG-CoA reductase --- p.7 / Chapter 1.3.2 --- HMG-CoA reductase as the rate-limiting enzyme in cholesterol synthesis --- p.11 / Chapter 1.3.3 --- LDL-receptor as the major protein removing plasma cholesterol …… --- p.12 / Chapter 1.3.4 --- LXR-α as an activator of CYP7A1 --- p.14 / Chapter 1.3.5 --- CYP7A1 controls the classical pathway for the elimination of hepatic cholesterol --- p.16 / Chapter 1.3.6 --- Bile acids as the metabolites of CYP7A1 --- p.17 / Chapter 1.4 --- Previous works in our laboratory --- p.20 / Chapter 1.5 --- Objective of this project --- p.22 / Chapter CHAPTER 2: --- INCREASED EXPRESSION OF LDL-RECEPTOR IS RESPONSIBLE FOR THE HYPO-RESPONSIVENESS OF RATS TO DIETARY CHOLESTEROL --- p.23 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Objective --- p.24 / Chapter 2.3 --- Methods and materials --- p.25 / Chapter 2.3.1 --- Animals --- p.25 / Chapter 2.3.2 --- Diets --- p.25 / Chapter 2.3.3 --- Determination of serum cholesterol --- p.26 / Chapter 2.3.4 --- Western blot --- p.26 / Chapter 2.3.5 --- Probe production for LDL-receptor --- p.27 / Chapter 2.3.5.1 --- Extraction of total RNA --- p.27 / Chapter 2.3.5.2 --- Reverse-transcription reaction of total RNA --- p.28 / Chapter 2.3.5.3 --- Polymerase chain reaction (PCR) of LDL- receptor fragment from cDNA template --- p.28 / Chapter 2.3.5.4 --- Separation and purification of PCR products --- p.29 / Chapter 2.3.5.5 --- Polishing of purified PCR products --- p.29 / Chapter 2.3.5.6 --- Ligation of PCR products and pPCR-script Amp SK(+) cloning vector --- p.30 / Chapter 2.3.5.7 --- Transformation --- p.30 / Chapter 2.3.5.8 --- Preparing glycerol stocks containing the bacterial clones --- p.31 / Chapter 2.3.5.9 --- Plasmid DNA preparation --- p.31 / Chapter 2.3.5.10 --- Clones confirmation by restriction enzyme digestion --- p.32 / Chapter 2.3.5.11 --- Clones confirmation by automatic sequencing --- p.32 / Chapter 2.3.5.12 --- Linearization of the plasmid DNA --- p.33 / Chapter 2.3.5.13 --- DIG-labeling of RNA probe --- p.35 / Chapter 2.3.5.14 --- Testing of DIG-labeled probe --- p.35 / Chapter 2.3.6 --- Probe production for HMG-CoA reductase --- p.36 / Chapter 2.3.7 --- Probe production for GAPDH --- p.37 / Chapter 2.3.8 --- Northern blot --- p.38 / Chapter 2.3.9 --- Determination of hepatic cholesterol --- p.39 / Chapter 2.3.10 --- Statistics --- p.40 / Chapter 2.4 --- Results --- p.42 / Chapter 2.4.1 --- Growth and food intake --- p.42 / Chapter 2.4.2 --- Effect of cholesterol supplements on serum cholesterol --- p.42 / Chapter 2.4.3 --- Effect of cholesterol supplements on liver cholesterol content --- p.45 / Chapter 2.4.4 --- "Stimulatory effect of high cholesterol diet on nSREBP-2, LDL-receptor and HMG-CoA reductase in rats" --- p.45 / Chapter 2.4.5 --- "Effect of high cholesterol diet on nSREBP-2, LDL-receptor and HMG-CoA reductase in hamsters" --- p.49 / Chapter 2.4.6 --- The regulation of LDL-receptor and HMG-CoA reductase existed at transcriptional level --- p.54 / Chapter 2.5 --- Discussion --- p.59 / Chapter CHAPTER 3: --- RATS ARE HYPO-RESPONSIVE TO DIETARY CHOLESTEROL DUE TO EFFICIENT ELIMINATION OF CHOLESTEROL --- p.67 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.2 --- Objective --- p.69 / Chapter 3.3 --- Methods and materials --- p.70 / Chapter 3.3.1 --- Animals and diets --- p.70 / Chapter 3.3.2 --- Western blot --- p.70 / Chapter 3.3.3 --- Probe production for CYP7A1 and LXR-α --- p.71 / Chapter 3.3.4 --- Northern blot --- p.71 / Chapter 3.3.5 --- Determination of fecal neutral and acidic sterols --- p.71 / Chapter 3.3.5.1 --- Separation of neutral and acidic sterols --- p.71 / Chapter 3.3.5.2 --- Neutral sterols analysis --- p.72 / Chapter 3.3.5.3 --- Acidic sterols analysis --- p.72 / Chapter 3.3.5.4 --- GLC analysis of neutral and acidic sterols --- p.73 / Chapter 3.3.6 --- Statistics --- p.73 / Chapter 3.4 --- Results --- p.76 / Chapter 3.4.1 --- Effect of cholesterol supplements on fecal total neutral sterols --- p.76 / Chapter 3.4.2 --- Effect of cholesterol supplements on fecal total bile acids --- p.76 / Chapter 3.4.3 --- CYP7A1 protein on rats showed a concentration-dependent increase with response to dietary cholesterol while hamsters did not --- p.79 / Chapter 3.4.4 --- The regulation of CYP7A1 was at transcriptional level --- p.79 / Chapter 3.4.5 --- LXR-α demonstrated a parallel changes in its expression at both translational and transcriptional level --- p.84 / Chapter 3.5 --- Discussion --- p.88 / Chapter CHAPTER 4: --- MECHANISM FOR INDIVIDUAL VARIATION OF SERUM CHOLESTEROL LEVEL IN RATS AND HAMSTERS FED A HIGH CHOLESTEROL DIET --- p.94 / Chapter 4.1 --- Introduction --- p.94 / Chapter 4.2 --- Objective --- p.96 / Chapter 4.3 --- Methods and materials --- p.97 / Chapter 4.3.1 --- Diet and animals --- p.97 / Chapter 4.3.2 --- Western blot --- p.97 / Chapter 4.3.3 --- Statistics --- p.97 / Chapter 4.4 --- Results --- p.99 / Chapter 4.4.1 --- Growth and food intake --- p.99 / Chapter 4.4.2 --- Change of serum cholesterol --- p.99 / Chapter 4.4.3 --- Correlation between various protein expression and serum cholesterol --- p.99 / Chapter 4.4.3.1 --- Correlation between LDL-receptor and serum total cholesterol in rats --- p.99 / Chapter 4.4.3.2 --- Correlation between CYP7A1 and serum total cholesterolin rats --- p.99 / Chapter 4.4.3.3 --- Correlation between nSREBP-2 and serum total cholesterolin rats --- p.105 / Chapter 4.4.3.4 --- Correlation between LXR-a and serum total cholesterol in rats --- p.105 / Chapter 4.4.3.5 --- Correlation between HMG-CoA reductase and serum total cholesterol in rats --- p.105 / Chapter 4.4.3.6 --- Correlation between LDL-receptor and serum total cholesterol in hamsters --- p.105 / Chapter 4.4.3.7 --- Correlation between CYP7A1 and serum total cholesterolin hamsters --- p.109 / Chapter 4.4.3.8 --- Correlation between nSREBP-2 and serum total cholesterolin hamsters --- p.109 / Chapter 4.4.3.9 --- Correlation between HMG-CoA reductase and serum total cholesterol in hamsters --- p.109 / Chapter 4.5 --- Discussion --- p.114 / Chapter CHAPTER 5: --- CONCLUSION --- p.117 / REFERENCES --- p.121

The temperature-dependent catalytic activity of rat liver 3-hydroxy-3 -methylglutaryl coenzyme A reductase (HMG-CoA reductase) displays the nonlinear Arrhenius behavior characteristic of many membrane-bound enzymes. A two-conformer equilibrium model has been developed to characterize this behavior. In the model, HMG-CoA reductase undergoes a conformational change from a low specific activity to a high specific activity form. This conformation change is apparently driven by a temperature-dependent phase transition of the membrane lipids. It has been found that this model accurately describes the data from diets including rat chow, low-fat, high-carbohydrate, and diets supplemented with fat, cholesterol or cholestyramine. The effects characterized by the model are consistent with the regulation of HMG-CoA reductase by enzyme-lipid interactions.

Introdução: As proteínas alimentares são fontes de peptídeos atuantes em vários processos metabólicos. Existem evidências de que a proteína do feijão caupi (Vigna unguiculata L. Walp) é capaz de reduzir o colesterol em hamsters e em humanos, porém, a permeabilidade após a digestão, o mecanismo de ação e a evidência direta da participação de peptídeos no metabolismo colesterol não são claros. Objetivo: Investigar a permeabilidade intestinal e avaliar o efeito nas vias luminal e endógena do metabolismo do colesterol de peptídeos provenientes de feijão caupi (Vigna unguiculata L. Walp). Metodologia: A permeabilidade dos peptídeos provenientes do caupi produzidos por hidrólise enzimática foi testada em linhagens de células Caco-2 usando placas Transwell®. Para investigar o efeito na via luminal, três peptídeos identificados na fração 3 kDa do hidrolisado (LLNPDDEQL; FFFGQDGGSKGEE e LNL) foram testados na solubilização de colesterol e fosfatidilcolina, no tamanho das micelas de colesterol e interação com ácidos biliares in vitro. Para verificar o efeito no metabolismo endógeno, linhagens de células HepG2 foram incubadas com peptídeos sintéticos (MELNAVSVVHS e MELNAVSVVSH) identificados como resultado do ensaio de permeação nas células Caco-2. A expressão de RNAm dos transportadores de colesterol NPC1L1, ABCA1 e ABCG1 foi realizada nas células Caco-2 e a expressão de HMGCR, SREBP2, LDLR, LXR, AMPK1 foi avaliada nas células HepG2. Resultados: A exposição das células Caco-2 à fração 3 kDa do hidrolisado (2,5 e 5 mg/mL) aumentou a expressão de ABCG1 nos tempos 6 h e 12 h. O níveis de RNAm dos genes SREBP2, HMGCR e LDLR reduziram nas HepG2 após 24h do tratamento com o peptídeo MELNAVSVVHS (50 M e 100 M). A fração 3 kDa do hidrolisado e os peptídeos LLNPDDEQL; FFFGQDGGSKGEE e LNL foram capazes de reduzir a solubilidade do colesterol micelar in vitro em no máximo 42 por cento , bem como, provocaram mudanças estruturais ao interagirem com a fosfatidilcolina, com destaque ao peptídeo LNL (50 por cento de ligação). O peptídeo LNL foi o único capaz de promover a precipitação do colesterol em forma de cristais devido à interação com os ácidos biliares. Conclusões: A fração 3 kDa do hidrolisado e todos os peptídeos testados foram capazes de insolubilizar o colesterol in vitro. Constata-se que o mecanismo de competição pelo espaço intramicelar com o colesterol se dá pela interação com os componentes micelares e não diretamente com o colesterol. O peptídeo do feijão caupi MELNAVSVVHS foi permeável e foi capaz de reduzir a expressão do fator de transcrição SREBP2 (consequentemente reduzindo HMGCR e LDLR) / Introduction: Food proteins are sources of peptides acting in sevral metabolic processes. There is evidence that cowpea (Vigna unguiculata L. Walp) protein is able to lower cholesterol levels in hamsters and humans, but its permeability after digestion, mechanism of action and direct evidences of peptide participation in cholesterol metabolism are not clear. Objective: To investigate the intestinal permeability and to evaluate the effect on the luminal and endogenous cholesterol metabolism pathways of peptides from cowpea (Vigna unguiculata L. Walp). Methods: The permeability of the cowpea peptides produced by enzymatic hydrolysis was tested on Caco-2 cell lines using Transwell® plates. To investigate the effect on the luminal pathway, three peptides identified in the 3 kDa hydrolyzate fraction (LLNPDDEQL; FFFGQDGGSKGEE and LNL) were tested for in vitro cholesterol and phosphatidylcholine solubilization, cholesterol micelle size changing and interaction with bile acids. To verify the effect on endogenous metabolism, HepG2 cell lines were incubated with synthetic peptides (MELNAVSVVHS or MELNAVSVVSH) identified as a result of the permeation assay on Caco-2 cells. The mRNA expression of the cholesterol transporters NPC1L1, ABCA1 and ABCG1 was performed on Caco-2 cells and the expression of HMGCR, SREBP2, LDLR, LXR, AMPK1 was evaluated in HepG2 cells. Results: Exposure of Caco-2 cells to the 3 kDa hydrolyzate fraction (2.5 and 5 mg/mL) increased ABCG1 expression at 6 h and 12 h times. The mRNA levels of the SREBP2, HMGCR and LDLR genes were reduced in HepG2 after 24h of treatment with the MELNAVSVVHS peptide (50 M and 100 M). The 3 kDa of the hydrolyzate fraction and the peptides LLNPDDEQL; FFFGQDGGSKGEE and LNL were able to reduce the solubility of micellar cholesterol in vitro in a maximum of 42 per cent , as well as, caused structural changes when interacting with phosphatidylcholine, with emphasis on the LNL peptide (50 per cent of binding). The LNL peptide alone was able to promote cholesterol precipitation in the form of crystals due to interaction with bile acids. Conclusions: The 3 kDa hydrolysate fraction and all peptides tested were able to insolubilize cholesterol in vitro. It was observed that the mechanism of competition for the intramicellar space with cholesterol is given by the interaction with the micellar components and not directly with the cholesterol. The MELNAVSVVHS cowpea peptide was permeable and was able to reduce the expression of the SREBP2 transcription factor (thereby reducing HMGCR and LDLR)

Introdução: As proteínas alimentares são fontes de peptídeos atuantes em vários processos metabólicos. Existem evidências de que a proteína do feijão caupi (Vigna unguiculata L. Walp) é capaz de reduzir o colesterol em hamsters e em humanos, porém, a permeabilidade após a digestão, o mecanismo de ação e a evidência direta da participação de peptídeos no metabolismo colesterol não são claros. Objetivo: Investigar a permeabilidade intestinal e avaliar o efeito nas vias luminal e endógena do metabolismo do colesterol de peptídeos provenientes de feijão caupi (Vigna unguiculata L. Walp). Metodologia: A permeabilidade dos peptídeos provenientes do caupi produzidos por hidrólise enzimática foi testada em linhagens de células Caco-2 usando placas Transwell®. Para investigar o efeito na via luminal, três peptídeos identificados na fração 3 kDa do hidrolisado (LLNPDDEQL; FFFGQDGGSKGEE e LNL) foram testados na solubilização de colesterol e fosfatidilcolina, no tamanho das micelas de colesterol e interação com ácidos biliares in vitro. Para verificar o efeito no metabolismo endógeno, linhagens de células HepG2 foram incubadas com peptídeos sintéticos (MELNAVSVVHS e MELNAVSVVSH) identificados como resultado do ensaio de permeação nas células Caco-2. A expressão de RNAm dos transportadores de colesterol NPC1L1, ABCA1 e ABCG1 foi realizada nas células Caco-2 e a expressão de HMGCR, SREBP2, LDLR, LXR, AMPK1 foi avaliada nas células HepG2. Resultados: A exposição das células Caco-2 à fração 3 kDa do hidrolisado (2,5 e 5 mg/mL) aumentou a expressão de ABCG1 nos tempos 6 h e 12 h. O níveis de RNAm dos genes SREBP2, HMGCR e LDLR reduziram nas HepG2 após 24h do tratamento com o peptídeo MELNAVSVVHS (50 M e 100 M). A fração 3 kDa do hidrolisado e os peptídeos LLNPDDEQL; FFFGQDGGSKGEE e LNL foram capazes de reduzir a solubilidade do colesterol micelar in vitro em no máximo 42 por cento , bem como, provocaram mudanças estruturais ao interagirem com a fosfatidilcolina, com destaque ao peptídeo LNL (50 por cento de ligação). O peptídeo LNL foi o único capaz de promover a precipitação do colesterol em forma de cristais devido à interação com os ácidos biliares. Conclusões: A fração 3 kDa do hidrolisado e todos os peptídeos testados foram capazes de insolubilizar o colesterol in vitro. Constata-se que o mecanismo de competição pelo espaço intramicelar com o colesterol se dá pela interação com os componentes micelares e não diretamente com o colesterol. O peptídeo do feijão caupi MELNAVSVVHS foi permeável e foi capaz de reduzir a expressão do fator de transcrição SREBP2 (consequentemente reduzindo HMGCR e LDLR) / Introduction: Food proteins are sources of peptides acting in sevral metabolic processes. There is evidence that cowpea (Vigna unguiculata L. Walp) protein is able to lower cholesterol levels in hamsters and humans, but its permeability after digestion, mechanism of action and direct evidences of peptide participation in cholesterol metabolism are not clear. Objective: To investigate the intestinal permeability and to evaluate the effect on the luminal and endogenous cholesterol metabolism pathways of peptides from cowpea (Vigna unguiculata L. Walp). Methods: The permeability of the cowpea peptides produced by enzymatic hydrolysis was tested on Caco-2 cell lines using Transwell® plates. To investigate the effect on the luminal pathway, three peptides identified in the 3 kDa hydrolyzate fraction (LLNPDDEQL; FFFGQDGGSKGEE and LNL) were tested for in vitro cholesterol and phosphatidylcholine solubilization, cholesterol micelle size changing and interaction with bile acids. To verify the effect on endogenous metabolism, HepG2 cell lines were incubated with synthetic peptides (MELNAVSVVHS or MELNAVSVVSH) identified as a result of the permeation assay on Caco-2 cells. The mRNA expression of the cholesterol transporters NPC1L1, ABCA1 and ABCG1 was performed on Caco-2 cells and the expression of HMGCR, SREBP2, LDLR, LXR, AMPK1 was evaluated in HepG2 cells. Results: Exposure of Caco-2 cells to the 3 kDa hydrolyzate fraction (2.5 and 5 mg/mL) increased ABCG1 expression at 6 h and 12 h times. The mRNA levels of the SREBP2, HMGCR and LDLR genes were reduced in HepG2 after 24h of treatment with the MELNAVSVVHS peptide (50 M and 100 M). The 3 kDa of the hydrolyzate fraction and the peptides LLNPDDEQL; FFFGQDGGSKGEE and LNL were able to reduce the solubility of micellar cholesterol in vitro in a maximum of 42 per cent , as well as, caused structural changes when interacting with phosphatidylcholine, with emphasis on the LNL peptide (50 per cent of binding). The LNL peptide alone was able to promote cholesterol precipitation in the form of crystals due to interaction with bile acids. Conclusions: The 3 kDa hydrolysate fraction and all peptides tested were able to insolubilize cholesterol in vitro. It was observed that the mechanism of competition for the intramicellar space with cholesterol is given by the interaction with the micellar components and not directly with the cholesterol. The MELNAVSVVHS cowpea peptide was permeable and was able to reduce the expression of the SREBP2 transcription factor (thereby reducing HMGCR and LDLR)

Plasma total, low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol concentrations increase immediately following birth. Interestingly, this increase is greater in breast-fed infants than in infants fed formula. The reason(s) why there are differences in plasma cholesterol concentrations between breast-fed and formula-fed infants is not known. However, this difference may be a consequence of the variations in lipid composition between milk and infant formula. Little is known regarding the specific effects of the lipid component(s) of infant diets on the expression of genes involved in hepatic lipid metabolism. The studies presented in this thesis determined whether the addition of cholesterol, arachidonic acid [20:4(n-6)] and docosahexaenoic acid [22:6(n-3)] to formula, and the positional distribution of fatty acids in formula triglycerides increases plasma cholesterol in formula-fed piglets to levels observed in milk-fed piglets. In study #1, piglets were fed from birth to 18 days of age with either a conventional infant formula (conventional formula) or a formula with synthesized triglycerides (TG) (synthesized TG formula). The conventional infant formula had 70% of the total 16:0, representing 23% of total fatty acids, esterified at the sn-1 and 3 positions of the formula triglyceride. The synthesized TG formula contained a similar percentage of 16:0, representing 23% of total fatty acids, but had 47% of the total 16:0 esterified at the centre (sn-2) position of the formula triglyceride. Each of the conventional and synthesized TG formulae were provided either without (<0.10 mM) or with 0.65mM cholesterol added to formula, 0.52mmol/L as unesterified cholesterol and 0.13 mmol/L as cholesterol oleate. A reference group of piglets was also fed sow milk. In study #1, the levels of hepatic HMG-CoA reductase mRNA, 7-a-hydroxylase (C7H) mRNA, and acetyl CoA carboxylase (ACC) mRNA were higher in the formula-fed than milk-fed piglets, irrespective of the formula cholesterol content or the positional distribution of fatty acids in the formula triglyceride. This was accompanied by lower plasma total and HDL cholesterol concentrations, lower hepatic triglyceride concentrations and lower concentrations of bile acids, cholesterol and phospholipid in bile of the formula-fed than milk-fed piglets. Adding cholesterol to the formula increased hepatic cholesterol concentrations and decreased hepatic levels of fatty acid synthase (FAS) mRNA, but had no effect on the plasma cholesterol concentrations of the formula-fed piglets. Directing 16:0 to the sn-2 position of the formula triglyceride led to lower plasma total cholesterol and triglyceride concentrations, lower concentrations of bile acids in bile, lower hepatic levels of FAS mRNA and activity, and higher hepatic levels of ACC mRNA than in piglets fed the conventional formula. In study #2, piglets were fed the conventional formula either without or with egg phospholipid (9.5g/L) to provide 0.8% 20:4(n-6) and 0.3% 22:6(n-3) of total fatty acids, or sow milk from birth to 15 days of age. Supplementing the conventional formula with egg phospholipid resulted in higher levels of 20:4(n-6) and 22:6(n-3) in liver and bile phospholipid, higher plasma HDL concentrations, higher bile acid and phospholipid concentrations in bile and lower hepatic ACC mRNA levels in the formula-fed piglets. The levels of 20:4(n-6) and 22:6(n-4) in liver and bile phospholipid were also higher in the piglets fed the supplemented formula than in the piglets fed milk. A significant inverse relation was found between the levels of hepatic ACC mRNA and the percentage of 20:4(n-6) in liver triglyceride and the percentage of 22:6(n-3) in liver phospholipid. Egg phospholipid supplementation of formula had no effect on hepatic LDL receptor mRNA or hepatic FAS activity and mRNA in the formula-fed piglets. The piglets fed either the supplemented or the conventional formula had lower levels of plasma cholesterol and higher levels of hepatic HMG-CoA reductase activity and mRNA and C7H mRNA than piglets fed milk. These studies show that early diet, that is, milk compared to formula feeding, results in lower levels of hepatic HMG-CoA reductase activity and mRNA and C7H mRNA accompanied by higher plasma cholesterol concentrations in piglets. Supplementing formula with cholesterol or the preferential esterification of 16:0 at the sn-2 position of the formula triglyceride did not raise plasma cholesterol concentrations and had no effect on hepatic HMG-CoA reductase activity and mRNA or C7H mRNA in formula-fed piglets. Supplementing formula with egg phospholipid, increased bile and liver phospholipid 20:4(n-6) and 22:6(n-3), decreased the levels of hepatic ACC mRNA and increased the concentrations of bile acids and phospholipid in bile. These findings suggest that milk-fed piglets have lower rates of hepatic cholesterol synthesis, lower rates of conversion of cholesterol to bile acids and the lipid present in sow milk and formula may be metabolized differently. These findings are significant in that they raise the question as to whether or not this effect of early diet will continue through to adulthood and influence metabolic response to diet fat.

The cholesterol-lowering effect of corn fiber oil, obtained from the seed coats of corn kernels, has been reported previously. Corn fiber oil contains phytosteryl fatty acyl esters, ferulate phytostanyl esters, and free phytosterols. To date, however, no studies have examined the cholesterol-lowering efficacy of ferulate phytostanyl esters. Moreover, although plant stanols and sterols have been established as cholesterol-lowering agents over the past five decades, their exact mechanisms of action are not clearly understood. One of the possible mechanism is that plant sterols/stanols disrupts the normal sub-cellular cholesterol absorption by down-regulation of the influx sterol transporters such as the Niemann pick C1 like 1(NPC1L1) protein and/or up-regulation of efflux sterol transporters such as the ATP binding cassette (ABC) G5 and ABCG8 protein. Hence, the objectives of this thesis were to assess the efficacy of corn fiber oil, ferulate phytostanyl esters and their parent compounds including sitostanol and ferulic acid, on plasma cholesterol levels. Further, objectives were to investigate their impact on parameters of cholesterol kinetics and gene expression of sterol transporters to obtain insight into their role in genetic control of regulation of cholesterol flux. Results of this experiment demonstrate that the hypocholesterolemic effect of corn fiber oil is mostly due to sitostanol, while esterification of ferulic acid and sitostanol yields no apparent synergistic cholesterol lowering effect. Present data exhibited a cholesterol absorption lowering effect of corn fiber oil and sitostanol and suggest that this effect may be due to up-regulation of intestinal enterocyte efflux sterol transporters such as ABCG5 and ABCG8.

Glial-derived lipoparticles facilitate the transport of cholesterol and lipids between cells within the CNS and have been shown to support neuronal growth and synaptogenesis. Partial deafferentation of the hippocampus by unilateral entorhinal cortex lesioning (uECL) induces well-described cytoarchitectural reorganisation and reactive sprouting in the dentate gyrus (DG). Previous studies have demonstrated a dynamic regulation of cholesterol homeostasis in the hippocampus following deafferentation, and suggest that mechanisms facilitating cholesterol transport are important during reinnervation. Furthermore, there is growing evidence that statins, a family of cholesterol-lowering drugs which inhibit the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA-R), may confer neuroprotection following trauma. / The ATP binding cassette transporters (ABC) A1 and G1 assist the generation of lipoparticles by mediating cholesterol and phospholipid efflux to extracellular apolipoprotein E (APOE), the brain's primary lipoprotein. To examine a role for these transporters in the regulation of cholesterol efflux during synaptic remodelling, and the effects of low-dose pravastatin (a potent HMGCoA-R inhibitor) on such intercellular transport mechanisms, we measured the expression of ABCA1, ABCG1, APOE, apoE(LDL)R and HMGCoA-R in the hippocampus of saline and pravastatin treated mice over time following uECL. It is shown here that ABCA1 and not ABCG1 is up-regulated at the level of mRNA and protein expression, along with APOE, in the hippocampus during active regeneration (14DPL) as determined by histochemical analysis of acetylcholinesterase staining density in the DG. While pravastatin treatment was observed to differentially influence the expression of ABCA1 mRNA and protein over time, no effects on APOE or ABCG1 mRNA expression were observed following uECL. Additionally, HMGCoA-R mRNA expression was significantly down-regulated at 21 DPL in the deafferented hippocampus in pravastatin-treated animals. While the low-dose pravastatin treatment applied here was sufficient to inhibit HMGCoA-R activity in the liver, enzymatic activity was unaffected in the cortex. / These findings suggest that ABCA1 and not ABCG1 may be important in the APOE-mediated cholesterol recycling observed during the active phase of neural reinnervation in response to uECL. In addition, the results presented here suggest that the administration of clinically-relevant statin therapy may be sufficient to influence the regulation of cerebral cholesterol homeostasis following trauma in the adult mouse brain.

Plasma total, low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol concentrations increase immediately following birth. Interestingly, this increase is greater in breast-fed infants than in infants fed formula. The reason(s) why there are differences in plasma cholesterol concentrations between breast-fed and formula-fed infants is not known. However, this difference may be a consequence of the variations in lipid composition between milk and infant formula. Little is known regarding the specific effects of the lipid component(s) of infant diets on the expression of genes involved in hepatic lipid metabolism. The studies presented in this thesis determined whether the addition of cholesterol, arachidonic acid [20:4(n-6)] and docosahexaenoic acid [22:6(n-3)] to formula, and the positional distribution of fatty acids in formula triglycerides increases plasma cholesterol in formula-fed piglets to levels observed in milk-fed piglets. In study #1, piglets were fed from birth to 18 days of age with either a conventional infant formula (conventional formula) or a formula with synthesized triglycerides (TG) (synthesized TG formula). The conventional infant formula had 70% of the total 16:0, representing 23% of total fatty acids, esterified at the sn-1 and 3 positions of the formula triglyceride. The synthesized TG formula contained a similar percentage of 16:0, representing 23% of total fatty acids, but had 47% of the total 16:0 esterified at the centre (sn-2) position of the formula triglyceride. Each of the conventional and synthesized TG formulae were provided either without (<0.10 mM) or with 0.65mM cholesterol added to formula, 0.52mmol/L as unesterified cholesterol and 0.13 mmol/L as cholesterol oleate. A reference group of piglets was also fed sow milk. In study #1, the levels of hepatic HMG-CoA reductase mRNA, 7-a-hydroxylase (C7H) mRNA, and acetyl CoA carboxylase (ACC) mRNA were higher in the formula-fed than milk-fed piglets, irrespective of the formula cholesterol content or the positional distribution of fatty acids in the formula triglyceride. This was accompanied by lower plasma total and HDL cholesterol concentrations, lower hepatic triglyceride concentrations and lower concentrations of bile acids, cholesterol and phospholipid in bile of the formula-fed than milk-fed piglets. Adding cholesterol to the formula increased hepatic cholesterol concentrations and decreased hepatic levels of fatty acid synthase (FAS) mRNA, but had no effect on the plasma cholesterol concentrations of the formula-fed piglets. Directing 16:0 to the sn-2 position of the formula triglyceride led to lower plasma total cholesterol and triglyceride concentrations, lower concentrations of bile acids in bile, lower hepatic levels of FAS mRNA and activity, and higher hepatic levels of ACC mRNA than in piglets fed the conventional formula. In study #2, piglets were fed the conventional formula either without or with egg phospholipid (9.5g/L) to provide 0.8% 20:4(n-6) and 0.3% 22:6(n-3) of total fatty acids, or sow milk from birth to 15 days of age. Supplementing the conventional formula with egg phospholipid resulted in higher levels of 20:4(n-6) and 22:6(n-3) in liver and bile phospholipid, higher plasma HDL concentrations, higher bile acid and phospholipid concentrations in bile and lower hepatic ACC mRNA levels in the formula-fed piglets. The levels of 20:4(n-6) and 22:6(n-4) in liver and bile phospholipid were also higher in the piglets fed the supplemented formula than in the piglets fed milk. A significant inverse relation was found between the levels of hepatic ACC mRNA and the percentage of 20:4(n-6) in liver triglyceride and the percentage of 22:6(n-3) in liver phospholipid. Egg phospholipid supplementation of formula had no effect on hepatic LDL receptor mRNA or hepatic FAS activity and mRNA in the formula-fed piglets. The piglets fed either the supplemented or the conventional formula had lower levels of plasma cholesterol and higher levels of hepatic HMG-CoA reductase activity and mRNA and C7H mRNA than piglets fed milk. These studies show that early diet, that is, milk compared to formula feeding, results in lower levels of hepatic HMG-CoA reductase activity and mRNA and C7H mRNA accompanied by higher plasma cholesterol concentrations in piglets. Supplementing formula with cholesterol or the preferential esterification of 16:0 at the sn-2 position of the formula triglyceride did not raise plasma cholesterol concentrations and had no effect on hepatic HMG-CoA reductase activity and mRNA or C7H mRNA in formula-fed piglets. Supplementing formula with egg phospholipid, increased bile and liver phospholipid 20:4(n-6) and 22:6(n-3), decreased the levels of hepatic ACC mRNA and increased the concentrations of bile acids and phospholipid in bile. These findings suggest that milk-fed piglets have lower rates of hepatic cholesterol synthesis, lower rates of conversion of cholesterol to bile acids and the lipid present in sow milk and formula may be metabolized differently. These findings are significant in that they raise the question as to whether or not this effect of early diet will continue through to adulthood and influence metabolic response to diet fat. / Graduate and Postdoctoral Studies / Graduate