This study was performed to determine the rates of in vitro cholesterol synthesis, as measured by ³H incorporation into cholesterol, in gluteal white adipose tissue and the intestine of infant rats during the early postnatal period. The reason for studying these parameters was to hopefully further elucidate the cause of the differences in blood cholesterol levels between rats raised in different litter sizes. Rats were raised in small (4/dam), medium (8/dam) and large (14/dam) litters. The rate of cholesterol synthesis in the proximal and distal region of the small intestine decreased from birth to a low 14 days later and then increased again by day 21. The rate of the decreases in cholesterol synthesis, from birth to the low on day 14, varied amongst the three litter sizes; the rate of synthesis in both regions of the intestine on day 7 was lowest in the larger litter, and was not significantly different from the values seen on day 14. Previous work has shown that plasma cholesterol levels were low in rats from large litters on day 7, thus in the early postnatal period low rates of intestinal cholesterol synthesis correlate with low plasma cholesterol values. After weaning, on day 21, the rate of synthesis in distal intestine was higher than that of proximal intestine in the medium and small litters, whereas the opposite was found in the larger litter. Cholesterol synthesis in gluteal white adipose tissue remained at a very low rate up to 21 days in the small and medium sized litters. In contrast, the rate of synthesis increased continuously in the large litter to nearly threefold the rate of that in the other two litter sizes on day 21. This study has shown that the rates of cholesterol synthesis in intestinal and adipose tissue can be altered during the early postnatal period by raising the rats in different litter sizes. Also, the pattern of development in the rates of cholesterol synthesis as measured by ³H incorporation into cholesterol are in general agreement with results reported for the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase for this same period in development. / Land and Food Systems, Faculty of / Graduate

The purpose of this study was to investigate whether increased endogenous cholesterol synthesis contributes to the elevated plasma cholesterol levels observed in type III hyperlipoproteinemia (type III HLP). Eight apolipoprotein (apo) E2 subjects with type III HLP and 8 apo E2 non-hyperlipidemic control subjects (controls) were given a priming bolus dose of deuterium oxide (D₂O) (0.7 g D₂O/kg body H2O). Daily M1 (central) pool free cholesterol fractional synthetic rate (FSR) was calculated as the incorporation rate of deuterium from body water into plasma free cholesterol. Blood samples were collected one half hour prior to, and at 12 hour intervals over 48 hours following, the bolus D₂O dose. Drinking water labelled at 1.4 and 0.7 g D₂O/liter H₂O was given on the fed and fasted days, respectively. Over 0-24 hours, subjects consumed a diet of three isocaloric meals which, in composition, approximated average North American intakes. Subjects fasted over 24-48 hours. The deuterium enrichment of plasma free cholesterol and plasma water was determined by isotope ratio mass spectrometry. When all subjects were included, mean (±SEM) free cholesterol overall FSR in type III HLPs (0.031 ± 0.006 per day) was not significantly different from controls (0.037 ± 0.004 per day). Estimated Ml total cholesterol pool size in type III HLPs (26.1 ± 1.9 g) and controls (24.9 ± 0.6 g) was not significantly different. When free cholesterol net synthesis was calculated as the absolute amount of cholesterol synthesized per day, based on Ml total cholesterol pool size, overall free cholesterol net synthesis in type III HLPs (0.304 ± 0.034 g/day) was not significantly different from controls (0.364 ± 0.035 g/day). When all subjects were included, overall free cholesterol FSR and overall free cholesterol net synthesis were significantly greater (p<0.001) in the fed (0.066 ± 0.006 day⁻¹ and 0.655 + 0.048 g/day, respectively) as compared to the fasted state (0.001 ± 0.004 day⁻¹ and 0.010 ± 0.037 g/day, respectively). In the fed state, type III HLPs tended to synthesize cholesterol at a lower rate and in a lower absolute amount as compared to controls, while the reverse was observed in the fasted state. These results suggest that: (1) the elevated plasma cholesterol levels observed in type III HLPs are not due to excess de novo cholesterol synthesis; (2) fasting significantly reduces cholesterol synthesis from the fed state. / Land and Food Systems, Faculty of / Graduate

To examine the effect of diet fat type on rates of cholesterol synthesis and esterification during feeding and fasting, nine healthy male subjects were fed solid-food diets of 40% fat as predominantly either olive oil (MONO), safflower-oil margarine (POLY), or butter (SAT). At the end of each two-week diet trial, subjects were given deuterium (D) oxide orally and de novo synthesis was measured from D incorporation into cholesterol and interpreted as rates of fractional synthesis (FSR) (pools/day) into the rapidly exchangeable free cholesterol (FC) pool. Absolute synthesis rates (ASR) were calculated as the product of FSR and the FC pool. Pool size for each subject was obtained from analysis of the specific activity decay curve of an intravenous injection of 4-14C-cholesterol over nine months. Synthesis was measured over two consecutive 12-h fed periods followed by two consecutive 12-h fasted periods. Serum samples were also assayed for lathosterol concentration, an index of cholesterol synthesis. Serum cholesterol and non-HDL cholesterol concentrations were highest on the SAT diet, lowest (P<0.001) on the POLY diet and intermediate on the MONO diet, triglyceride levels were greater (P<0.03) on the SAT diet than on the POLY diet, and HDL levels were lowest (P<0.05) on the SAT diet and highest on the MONO diet. Cholesterol D enrichment and FSR during each 12-h period were greater (P<0.014) on the POLY diet than on the SAT diet; MONO enrichment and FSR were not significantly different from those on the other two diets. Similar results were obtained for rates of cholesterol esterification (P<0.001). Deuterium enrichment data suggested, and lathosterol data confirmed, that free cholesterol synthesis was greater during the fed period than during the fasted period (P<0.01); however, this could not be confirmed for rates of cholesterol esterification. Results suggest that POLY fat feeding augments de novo cholesterol synthesis without adverse effects on total serum cholesterol concentrations, and that the deleterious effects of SAT fat on serum cholesterol are not brought about by augmented de novo synthesis. Finally, the combination of deuterium incorporation and mathematical modelling produces estimates of daily cholesterol synthesis which are compatible with those invoked by more laborious techniques.

To examine the effect of diet fat type on rates of cholesterol synthesis and esterification during feeding and fasting, nine healthy male subjects were fed solid-food diets of 40% fat as predominantly either olive oil (MONO), safflower-oil margarine (POLY), or butter (SAT). At the end of each two-week diet trial, subjects were given deuterium (D) oxide orally and de novo synthesis was measured from D incorporation into cholesterol and interpreted as rates of fractional synthesis (FSR) (pools/day) into the rapidly exchangeable free cholesterol (FC) pool. Absolute synthesis rates (ASR) were calculated as the product of FSR and the FC pool. Pool size for each subject was obtained from analysis of the specific activity decay curve of an intravenous injection of 4-14C-cholesterol over nine months. Synthesis was measured over two consecutive 12-h fed periods followed by two consecutive 12-h fasted periods. Serum samples were also assayed for lathosterol concentration, an index of cholesterol synthesis. Serum cholesterol and non-HDL cholesterol concentrations were highest on the SAT diet, lowest (P<0.001) on the POLY diet and intermediate on the MONO diet, triglyceride levels were greater (P<0.03) on the SAT diet than on the POLY diet, and HDL levels were lowest (P<0.05) on the SAT diet and highest on the MONO diet. Cholesterol D enrichment and FSR during each 12-h period were greater (P<0.014) on the POLY diet than on the SAT diet; MONO enrichment and FSR were not significantly different from those on the other two diets. Similar results were obtained for rates of cholesterol esterification (P<0.001). Deuterium enrichment data suggested, and lathosterol data confirmed, that free cholesterol synthesis was greater during the fed period than during the fasted period (P<0.01); however, this could not be confirmed for rates of cholesterol esterification. Results suggest that POLY fat feeding augments de novo cholesterol synthesis without adverse effects on total serum cholesterol concentrations, and that the deleterious effects of SAT fat on serum cholesterol are not brought about by augmented de novo synthesis. Finally, the combination of deuterium incorporation and mathematical modelling produces estimates of daily cholesterol synthesis which are compatible with those invoked by more laborious techniques. / Land and Food Systems, Faculty of / Graduate

No description available.

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, which catalyses the second committed step in cholesterol biosynthesis. The mechanism by which SPF stimulates this enzyme is not understood and the goal of these studies was to see if SPF affected cholesterol synthesis in cultured cells. Rat supernatant protein factor-like protein (SPF2) shares 77% sequence identity with human SPF. In my studies SPF2 also stimulated squalene monooxygenase in vitro and incubation of SPF2 with protein kinase A (PKA) and C increased its activity by about 2-fold, as shown earlier with SPF. GTP and GDP prevented the stimulation of squalene monooxygenase by SPF2, suggesting that binding of these nucleotides inhibits SPF2. This inhibition could be prevented by the addition of -tocopherol, although -tocopherol alone had no effect on SPF2 activity in vitro. Expression of human SPF in hepatoma cells, which lack expression of endogenous SPF, increased cholesterol synthesis by 2-fold and addition of dibuytrylcAMP, a PKA activator, to these cells yielded an additional 62% increase whereas addition of a PKA inhibitor completely blocked the ability of SPF to stimulate cholesterol synthesis. To further confirm a role for phosphorylation in the regulation of SPF, substitution of alanine for serine-289 (a putative PKA recognition site) reduced the PKA-mediated activation of SPF in vitro by 50%, as measured with microsomal squalene monooxygenase and completely blocked the ability of SPF to stimulate cholesterol synthesis in hepatoma cells. In further structure-function studies, deletion of the carboxy-terminal Golgi-dynamics domain greatly increased the ability of SPF to stimulate squalene monooxygenase in microsomes, but, paradoxically prevented SPF from stimulating cholesterol synthesis in cell culture. Addition of brefeldin A, which disrupts Golgi formation, also abolished the ability of SPF to stimulate cholesterol synthesis, supporting a role for the Golgi in SPF function. Since squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway was investigated. The substitution of 14Cmevalonate for 14C-acetate completely blocked an SPF-induced 1.5-fold increase in squalene synthesis, suggesting that SPF stimulated mevalonate synthesis at HMGCoA reductase. 2,3-Oxidosqualene synthesis from 14C-mevalonate remained elevated (1.3-fold) with mevalonate demonstrating that SPF also stimulated squalene monooxygenase in hepatoma cells. SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels in cells, indicating that SPF directly activated these enzymes. Indeed, addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and, coupled with the ability of PKA-mediated phosphorylation to regulate SPF activity, suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.

Elevated plasma lipids are risk factors for cardiovascular disease (CVD). In certain conditions plasma lipids are normal yet individuals experience increased morbidity. Type 1 diabetes (T1D) is associated with elevated CVD despite normal lipids, while in liver failure low plasma lipids may indicate increasing hepatic damage. Plasma lipids can therefore belie underlying dysregulated lipid metabolism. Islet (ITx) or liver (LTx) transplants represent therapies for T1D and liver failure, respectively, but are associated with altered lipid metabolism attributed to immunosuppressive medications; however, causative mechanisms are unknown. Partial success of dietary therapy in post-transplant patients may be due to interventions limited in scope. Regulation of plasma lipids involve absorption, synthesis, and clearance. These studies examined lipogenesis and cholesterol synthesis using deuterium incorporation. In brittle T1D lipogenesis and cholesterol synthesis were similar to healthy controls; however hepatic lipogenesis and cholesterol synthesis tended to be lower in T1D compared to matched control subjects. Plasma cholesterol was lower and triglyceride similar in liver failure patients compared to controls. Lipogenesis was higher while cholesterol synthesis was lower in liver failure compared to controls. Disturbances in lipid synthesis may be influenced by underlying disease, such as hepatitis C. In ITx and LTx lipogenesis was lower whereas cholesterol synthesis was similar compared to controls. Lipid synthesis is therefore unlikely to contribute to post-transplant hyperlipidemia, inviting investigation of other mechanisms. Dietary intervention emphasizing fish oil, phytosterols, soy, fibers, and almonds lowered plasma lipids in controls but had mixed effects in transplant subjects. Reduction in plasma lipids occurred in transplant patients with higher baseline lipids, suggesting this intervention may be successful in hyperlipidemic patients; however the potential of this diet intervention requires further study in hyperlipidemic patients. Diet intervention lowered lipogenesis but did not significantly change 24h cholesterol synthesis in controls. Diet did not change 24h lipogenesis or cholesterol synthesis in transplant subjects. Plasma lipid response to dietary therapy was related to baseline cholesterol synthesis and to dietary compliance in transplant subjects. Further study is required to determine if cholesterol synthesis is predictive of response to diet. / Nutrition and Metabolism

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths worldwide with a five-year survival rate of only 9%. The mevalonate (MVA) pathway, which results in synthesis of cholesterol, has been demonstrated to be overexpressed in numerous cancers, leading to its involvement in various aspects of the disease, including proliferation, invasion, and metastasis. In recent years, the MVA pathway has been implicated in PDAC as well, however the underlying mechanisms behind its upregulation in this context, as well as its role in tumor progression, remain largely unknown. An initial analysis of The Cancer Genome Atlas (TCGA) datasets using the Gene Expression Profiling Interactive Analysis (GEPIA) tool revealed one mevalonic gene in particular, SQLE—encoding squalene epoxidase (SQLE)—to be significantly overexpressed in PDAC compared to other genes within the pathway. We find that patients with higher SQLE expression profiles have significantly reduced five-year survival rates that can be further correlated to tumor stage and grade, and we demonstrate that knockdown of SQLE, a branchpoint enzyme of the cholesterol pathway, reduces tumor proliferation in both two-dimensional and three-dimensional assays. We further characterize the SQLE promoter landscape by demonstrating an interesting dynamic of gene activation between the sterol regulatory element binding protein-2 (SREBP2), BRG1-specific SWI/SNF chromatin remodelers, and mutant p53. These results highlight SQLE as a therapeutic target in PDAC and provide a better understanding of its regulation in the context of this devastating disease.

No description available.

The cholesterol-lowering effect of mixed linkage (1→3) (1→4)-β-D-glucans (β-glucan) from barley has been documented, yet the underlying mechanism responsible for this action and factors influencing it, such as physicochemical properties of β-glucan and genetic background of an individual, remain unclear.As a component of dietary fibre, β-glucan also has the potential to shift the gut microbial community, however, whether alterations in the gut microbiota are associated with the physiological effects of β-glucan have yet to be determined. This study was designed to assess the effects of β-glucan molecular weight (MW) and dose on loweringserum cholesterol levels and to elucidate its mechanism of action in human subjects. Additionally, this study examined gene-diet interactions as well as changes in the gut microbiota profile following consumption of barley foods. In a controlled four phase crossover trial, mildly hypercholesterolemic but otherwise healthy subjects (n =30) were randomly assigned to receive breakfasts containing 3g high MW (HMW), 5g low molecular weight (LMW), 3g LMW barley β-glucan or a control diet with wheat and rice (WR control), each for 5 weeks. The washout period between the phases was 4 weeks. The consumption of 3g/d HMW diet lowered total cholesterol (TC) compared with WR control diet (P =0.0046), but not the LMW diet at either 3g/d or 5g/d. Individuals with the SNP rs3808607-G allele of CYP7A1 had greater TC reduction in response to 3g/d HMW β-glucan diet compared to the individuals carrying homozygous TT alleles (P<0.01). Cholesterol absorption and synthesis were not changed, but bile acid synthesis increased by 3g/d HMW diet compared to the control. Consuming 3g HMW/d β-glucan altered gut microbiota at the phylum and genus levels and the impacted microbial members was correlated with favorable shifts of cardiovascular disease risk factors. In conclusion, physicochemical properties of β-glucan play critical roles in the cholesterol-lowering effect and gut microbiota alteration ability of β-glucan. The results suggest the increasing bile acid synthesis rather than inhibiting cholesterol absorption and synthesis is the mechanism responsible for the cholesterol reducing property of β-glucan.The altered microbiota profile by HMW β-glucan is associated with its physiological effect. / February 2016