Tesi sul tema "Cholesterol Physiological effect"

Statins are widely used to treat hypercholesterolemia. Statins inhibit cholesterol biosynthesis, thereby activating genes involved in cholesterol homeostasis, which are under the control of the Sterol Regulatory Element (SRE). Statins also have cholesterol-independent beneficial cardiovascular effects mediated through the phosphoinositide 3-kinase (PI3-K) / Akt signaling pathway and by inhibition of protein prenylation. Because statins inhibit the synthesis of isoprenoids, they can act by inhibiting the small signaling GTPases Ras and Rho, which require post-translational prenylation to become membrane-anchored and functional. We showed that simvastatin-mediated inhibition of protein prenylation does not appear to play a role in activation of SRE transcriptional activity in HepG2 cells. We also found that when isoprenoids were replenished, basal phospho-Akt decreased, suggesting that inhibition of prenylation by simvastatin mediates Akt phosphorylation. Future studies will be needed to investigate the role that inhibition of protein prenylation plays in the activation of the PI3-K/Akt pathway by simvastatin.<br>Department of Biology

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.

The objective of this study was to examine the effects of plant sterols and/or glucomannan on lipid profiles and cholesterol kinetics in hyperlipidemic individuals with or without type 2 diabetes. It was hypothesized that plant sterols and glucomannan reduce circulating cholesterol levels and may have an additive or synergistic effect when combined by reducing cholesterol absorption. Thirteen type 2 diabetics and sixteen non-diabetics all mildly hypercholesterolemic free living subjects participated in a randomized crossover trial consisting of 4 phases, 21 days each. Subjects consumed plant sterols and glucomannan during the trial. Overall reductions in total and LDL-cholesterol levels were greater (P<0.05) after consumption of the combination supplement. Effects of supplements were not different between diabetics and non-diabetics. No significant changes were observed in cholesterol absorption or synthesis in both diabetics and non-diabetics. The intake of plant sterols and glucomannan together may be an alternative approach in reducing blood cholesterol levels.

The objective of this research was to examine the effects of sitosterol and sitostanol supplementation on plasma cholesterol levels and cholesterol metabolism in hypercholesterolemic subjects consuming a fixed foods diet in a four-phase crossover design. It was hypothesized that addition of either phytosterols, phytostenols, or a 50:50 mixture of sterols and stanols to butter would reduce circulating cholesterol levels, despite butter's hypercholesterolemic effect, through actions involving cholesterol absorption, synthesis, and turnover rates. The data obtained indicate that in their free, unesterified form, plant sterols and stanols lower plasma LDL cholesterol equivalently in hypercholesterolemic subjects. Results of this study provide new data that phytosterols and stanols function by suppressing cholesterol absorption while increasing cholesterol synthesis, however, the de-suppression in synthesis cannot fully compensate for the decrease in absorption making the treatment effective, thus may assist in the development of a food which offers health-promoting advantages related to the prevention of cardiovascular disease.

Background. A high ratio of total cholesterol to high-density lipoprotein (HDL) cholesterol, in addition to increased levels of small low-density lipoprotein (LDL) particles, are important indicators of cardiovascular disease risk. Therefore, interventions that combine the lowering of total cholesterol and raising of HDL cholesterol concentrations that also increase LDL particle size, may be preventive against cardiovascular disease. Plant sterols decrease total cholesterol and LDL cholesterol levels by 10-15%, while exercise increases HDL cholesterol levels by 4-22%. In view of their complementary effects, combining plant sterols with exercise would appear to be an effective lifestyle therapy to decrease the risk of future cardiovascular disease.<br>Objective. The aim of this study was to examine the independent and combined effects of plant sterols and exercise on blood lipid levels, and LDL particle size in previously sedentary, hypercholesterolemic adults. An additional objective of this trial was to assess the underlying mechanism by which this combination therapy modulates whole body cholesterol metabolism, to in turn improve lipid profiles.<br>Methods. In an 8-week, parallel-arm trial, 84 subjects were randomized to 1 of 4 interventions: (1) plant sterols and exercise,(2) plant sterols alone, (3) exercise alone, or (4) control. Blood lipid concentrations were measured using enzymatic kits, and LDL particle size was assessed using polyacrylamide gel electrophoresis. Cholesterol absorption and synthesis were determined using the single isotope single tracer technique and the deuterium incorporation approach, respectively.<br>Results. Plant sterol supplementation decreased (P &lt; 0.01) total cholesterol concentrations by 8.2% when compared to baseline. Exercise increased (P &lt; 0.01) HDL cholesterol levels by 7.5% while decreasing (P &lt; 0.01) triglyceride concentrations by 13.3% when compared to baseline. Exercise reduced (P &lt; 0.05) post-treatment LDL peak particle size from 255 to 253 A, and decreased (P &lt; 0.05) the proportion of large LDL particles by 13.1%. Plant sterols had no effect on particle size distribution. Plant sterol supplementation decreased (P &lt; 0.01) intestinal cholesterol absorption by 18%, while exercise had no effect on cholesterol absorption. Non-significant increases in cholesterol synthesis rates of 63%, 59%, and 57%, were observed in the combination, exercise, and plant sterol groups, respectively, relative to control.<br>Conclusion. These findings suggest that this combination therapy yields the most favourable alterations in lipid profiles when compared to each intervention alone. This combined intervention exerts its beneficial effects on lipid profiles by suppressing intestinal cholesterol absorption. Therefore, this lifestyle therapy may be an effective means of decreasing the risk of cardiovascular disease in hypercholesterolemic adults.

The objective of this study was to compare the effect of phytosterols (PS) on lipid profiles and cholesterol kinetics of hypercholesterolemic individuals with or without type 2 diabetes. It was hypothesised that the response to PS would differ between both groups due to different lipid metabolism. During this randomised, double blind, crossover trial, participants consumed a controlled diet with placebo or PS for 21 days.<br>Plasma total cholesterol (TC) decreased with placebo and PS (10.9% and 9.7% in non-diabetic versus 11.6% and 13.6% in diabetic participants, p < 0.05). Plasma low-density lipoprotein cholesterol (LDL) significantly decreased with PS in both groups. The reduction in LDL with PS was greater in diabetic compared to non-diabetic individuals (29.8% versus 14.9%, p < 0.05). Cholesterol absorption decreased on average (p = 0.06) by 26.5% with PS compared with placebo in the diabetic group only. Therefore, a controlled heart healthy diet reduced TC and LDL concentrations in non-diabetic and diabetic individuals. Adding PS as adjuncts to a hypocholesterolemic dietary treatment was associated with lower LDL concentrations and cholesterol absorption in hypercholesterolemic participants with type 2 diabetes.

The purpose of the present study was to determine the acute effects of strenuous exercise on the following blood constituents: total cholesterol (TC) 1 triglycerides (TG) 1 high density lipoproteins (HDL-C) 1 and low density lipoproteins (LDL-C). A single case study was performed during a 20 day testing period. Two century bicycle rides ( 100 miles) were used as the strenuous exercise bouts. Blood samples were drawn each day and immediately after each 100 mile ride. A pre-set exercise and diet regimen were followed every day of the 20 day procedure. A t-test upon TC 1 TG 1 HDL-C 1 and LDL-C was done to determine the statistical significance between two 100 mile cycling rides and the training days. The change upon TC was an increase of 11.1mg/dl and the change upon TG was an increase of 66.8mg/dl. The t-tests upon both of these variables were found to be significant at the <0.05 level. The change upon HDL-C was an increase of 3.2mg/dl but a ttest showed no statistical significance at the <0.05 level. The change upon LDL-C was a decrease of 2.6mg/dl but a t-test showed no statistical significance at the <0.05 level. Total cholesterol to HDL-C ratio (TC/HDL-C) did not change and a t-test showed no statistical significance at the <0.05 level. The ratio stayed at 2.5 for the duration of the study period. It was determined that an acute bout of exercise significantly changed TC and TG levels. Total cholesterol mean values changed from 106.4mg/dl ± 1.11 to 117.5mg/dl ± 3. 53. Mean TG values changed from 66. 2mg/dl ± 4. 08 to 113mg/dl ± 16.97. The acute bout of exercise did not significantly change HDL-C or LDL-C. possibilities are discussed.

The purpose of this study was to determine the effectiveness of group nutrition and physical activity education classes in lowering cholesterol levels of worksite employees at 6-and 12-month intervals. A total of 32 participants were followed through the study. The group of participants included 24 female and 8 males, all over the age of 40. The data were analyzed using one-way ANOV A with repeated n1easures, post¬hoc analysis, Pearson's correlation coefficient, and ANCOV A to test six null hypotheses. Statistically significant differences in HDL cholesterol levels were found between baseline and 12 months (p=O.OOO) and between 6 months and 12 months (p=O.OOO). Statistically significant differences were also found in TCIHDL cholesterol ratios between baseline and 12 months (p=0.02) and between 6 months and 12 months (p=0.021).<br>Department of Family and Consumer Sciences

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.

Lam, Cheuk Kai.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.<br>Includes bibliographical references (leaves 148-173).<br>Abstracts in English and Chinese.<br>ACKNOWLEDGEMENTS --- p.i<br>ABSTRACT --- p.ii<br>LIST OF ABBREVIATIONS --- p.vi<br>TABLE OF CONTENTS --- p.x<br>Chapter CHAPTER 1 --- GENERAL INTRODUCTION<br>Chapter 1.1 --- Introduction to Cholesterol and Its Related Diseases --- p.1<br>Chapter 1.1.1 --- Chemistry of cholesterol --- p.1<br>Chapter 1.1.2 --- Physiological importance of cholesterol --- p.1<br>Chapter 1.1.3 --- Pathological effects of cholesterol --- p.3<br>Chapter 1.1.3.1 --- Mechanism of atherosclerosis --- p.3<br>Chapter 1.2 --- Cholesterol Homeostasis --- p.6<br>Chapter 1.2.1 --- Liver as the main organ for cholesterol metabolism --- p.6<br>Chapter 1.2.2 --- Regulatory sites of cholesterol metabolism --- p.6<br>Chapter 1.2.2.1 --- Regulation of cholesterol absorption by acyl coenzyme A: cholesterol acyltransferase (ACAT) --- p.6<br>Chapter 1.2.2.2 --- Sterol regulatory element-binding protein 2 (SREBP-2) as a transcription factor for 3 -hydroxy-3 -methylglutaryl coenzyme A reductase (HMGR) and low-density lipoprotein receptor (LDLR) --- p.10<br>Chapter 1.2.2.3 --- Roles ofLDLR --- p.11<br>Chapter 1.2.2.4 --- Rate limiting role of HMGR in cholesterol de novo synthesis --- p.14<br>Chapter 1.2.2.5 --- Roles of liver-X-receptor-a (LXR-a) in cholesterol catabolism --- p.16<br>Chapter 1.2.2.6 --- Roles of CYP7A1 in catabolism of cholesterol into bile acids --- p.19<br>Chapter 1.2.2.7 --- Roles of cholesterol ester transfer protein (CETP) in maintaining cholesterol distribution in blood --- p.22<br>Chapter CHAPTER 2 --- EFFECT OF OCTADECAENOIC ACIDS ON BLOOD CHOLESTEROL IN HAMSTERS<br>Chapter 2.1 --- Introduction --- p.25<br>Chapter 2.1.1 --- Effects of polyunsaturated fatty acids (PUFAs) on blood cholesterol --- p.25<br>Chapter 2.1.2 --- Differential effects of 18-C PUFAs on lowering blood cholesterol in vivo --- p.25<br>Chapter 2.1.3 --- "Structures, metabolism and conjugation of octadecaenoic acids (ODA)" --- p.26<br>Chapter 2.1.4 --- Objectives --- p.26<br>Chapter 2.2 --- Experiment 1 --- p.28<br>Chapter 2.2.1 --- Materials and methods --- p.28<br>Chapter 2.2.1.1 --- Experimental fatty acids --- p.28<br>Chapter 2.2.1.1.1 --- Isolation of LN from flaxseed --- p.28<br>Chapter 2.2.1.1.2 --- Isolation of CLN from tung seed --- p.28<br>Chapter 2.2.1.2 --- Animals --- p.29<br>Chapter 2.2.1.3 --- Diets --- p.30<br>Chapter 2.2.1.4 --- Plasma lipid measurements --- p.30<br>Chapter 2.2.1.5 --- Plasma CETP activity measurement --- p.30<br>Chapter 2.2.1.6 --- "Measurement of liver SREBP-2, LDLR, HMGR and CYP7A1 protein abundance by Western blotting" --- p.34<br>Chapter 2.2.1.7 --- "Measurement of hepatic SREBP-2, LDLR, HMGR, LXR, CYP7A1, CETP, SR-B1 and LCAT mRNA by real time PCR" --- p.35<br>Chapter 2.2.1.7.1 --- Extraction of mRNA --- p.35<br>Chapter 2.2.1.1.2 --- Complementary DNA synthesis --- p.36<br>Chapter 2.2.1.7.3 --- Real-time polymerase chain reaction (PCR) anaylsis --- p.36<br>Chapter 2.2.1.8 --- Determination of cholesterol in liver --- p.37<br>Chapter 2.2.1.9 --- Determination of fecal neutral and acidic sterols --- p.38<br>Chapter 2.2.1.9.1 --- Determination of fecal neutral sterols --- p.39<br>Chapter 2.2.1.9.2 --- Determination of fecal acidic sterols --- p.41<br>Chapter 2.2.1.10 --- Statistics --- p.43<br>Chapter 2.2.2 --- Results --- p.44<br>Chapter 2.2.2.1 --- Growth and food intake --- p.44<br>Chapter 2.2.2.2 --- Organ weights --- p.44<br>Chapter 2.2.2.3 --- "Effects of ODA on serum TC, TG and HDL-C" --- p.44<br>Chapter 2.2.2.4 --- Effect of ODA on liver cholesterol --- p.48<br>Chapter 2.2.2.5 --- Effect of ODA on fecal neutral sterol output --- p.48<br>Chapter 2.2.2.6 --- Effect of ODA on fecal acidic sterol output --- p.48<br>Chapter 2.2.2.7 --- Effect of ODA on cholesterol balance in hamsters --- p.52<br>Chapter 2.2.2.8 --- Effect of ODA on plasma CETP activity --- p.52<br>Chapter 2.2.2.9 --- Correlation between blood TC and liver cholesterol --- p.52<br>Chapter 2.2.2.10 --- Correlation between blood HDL-C and liver cholesterol --- p.52<br>Chapter 2.2.2.11 --- Correlation between blood nHDL/HDL ratio and liver cholesterol --- p.52<br>Chapter 2.2.2.12 --- Effect ofODA on liver SREBP-2 immunoreactive mass --- p.58<br>Chapter 2.2.2.13 --- Effect of ODA on liver LDLR immunoreactive mass --- p.58<br>Chapter 2.2.2.14 --- Effect of ODA on liver HMGR immunoreactive mass --- p.58<br>Chapter 2.2.2.15 --- Effect of ODA on liver LXR immunoreactive mass --- p.58<br>Chapter 2.2.2.16 --- Effect of ODA on liver CYP7A1 immunoreactive mass --- p.63<br>Chapter 2.2.2.17 --- Effects ofODA on hepatic CETP mRNA --- p.65<br>Chapter 2.2.2.18 --- Effects of ODA on hepatic LDLR mRNA --- p.65<br>Chapter 2.2.2.19 --- Effects of ODA on hepatic LXR mRNA --- p.65<br>Chapter 2.2.2.20 --- Effects of ODA on hepatic CYP7A1 mRNA --- p.65<br>Chapter 2.3 --- Experiment 2 --- p.70<br>Chapter 2.3.1 --- Materials and Methods --- p.70<br>Chapter 2.3.1.1 --- Experimental diets --- p.70<br>Chapter 2.3.1.2 --- Animals --- p.70<br>Chapter 2.3.1.3 --- Intestinal acyl coenzyme A: cholesterol acyltransferase (ACAT) activity measurement --- p.70<br>Chapter 2.3.1.3.1 --- Preparation of intestinal microsome --- p.71<br>Chapter 2.3.1.3.2 --- ACAT activity assay --- p.71<br>Chapter 2.3.2 --- Results --- p.73<br>Chapter 2.3.2.1 --- Growth and food intake --- p.73<br>Chapter 2.3.2.2 --- Organ weights --- p.73<br>Chapter 2.3.2.3 --- "Effect of ODA on serum TC, TG and HDL-C" --- p.73<br>Chapter 2.3.2.4 --- Effect of ODA feeding on fecal neutral sterol content --- p.77<br>Chapter 2.3.2.5 --- Effect of ODA feeding on fecal acidic sterol content --- p.77<br>Chapter 2.3.2.6 --- Effect of ODA feeding on intestinal acyl coenzyme A: acyl cholesterol transferase (ACAT) activity --- p.77<br>Chapter 2.4 --- Discussion --- p.81<br>Chapter CHAPTER 3 --- EFFECT OF OCTADECAENOIC ACIDS ON CHOLESTEROL-REGULATING GENES IN HepG2<br>Chapter 3.1 --- Introduction --- p.86<br>Chapter 3.1.1 --- HepG2 as a model of cholesterol regulation --- p.86<br>Chapter 3.1.2 --- Effect of polyunsaturated fatty acids (PUFAs) on cholesterol regulating genes in cultured cells --- p.87<br>Chapter 3.1.3 --- Objectives --- p.89<br>Chapter 3.2 --- Materials and Methods --- p.90<br>Chapter 3.2.1 --- Cell culture --- p.90<br>Chapter 3.2.2 --- "Measurement of SREBP-2, LDLR, HMGR and CYP7A1 protein abundance by Western blotting" --- p.92<br>Chapter 3.2.3 --- "Measurement of cellular SREBP-2, LDLR, HMGR, LXR, CYP7A1 and CETP mRNA by real time PCR" --- p.93<br>Chapter 3.2.4 --- Statistics --- p.93<br>Chapter 3.3 --- Results --- p.95<br>Chapter 3.3.1 --- Effect of ODA on HepG2 SREBP-2 immunoreactive mass --- p.95<br>Chapter 3.3.2 --- Effect of ODA on HepG2 HMGR immunoreactive mass --- p.95<br>Chapter 3.3.3 --- Effect of ODA on HepG2 LDLR immunoreactive mass --- p.95<br>Chapter 3.3.4 --- Effect of ODA on HepG2 LXR immunoreactive mass --- p.95<br>Chapter 3.3.5 --- Effect of ODA on HepG2 CYP7A1 immunoreactive mass --- p.96<br>Chapter 3.3.6 --- Effect of ODA supplementation on HepG2 SREBP-2 mRNA expression --- p.102<br>Chapter 3.3.7 --- Effect of ODA supplementation on HepG2 SREBP-2 mRNA expression --- p.102<br>Chapter 3.3.8 --- Effect of ODA supplementation on HepG2 LDLR mRNA expression --- p.102<br>Chapter 3.3.9 --- Effect of ODA supplementation on HepG2 LXR mRNA expression --- p.106<br>Chapter 3.3.10 --- Effect of ODA supplementation on HepG2 CYP7A1 mRNA expression --- p.106<br>Chapter 3.3.11 --- Effect of ODA supplementation on HepG2 CETP mRNA expression --- p.106<br>Chapter 3.4 --- Discussion --- p.110<br>Chapter CHAPTER 4 --- EFFECT OF APPLE POLYPHENOLS ON BLOOD CHOLESTEROL IN HAMSTERS<br>Chapter 4.1 --- Introduction --- p.114<br>Chapter 4.1.1 --- Apple is a commonly consumed fruit worldwide --- p.114<br>Chapter 4.1.2 --- Potential health effects of apples --- p.114<br>Chapter 4.1.3 --- Abundance of polyphenols in apple --- p.115<br>Chapter 4.1.4 --- Fuji variety of apple --- p.116<br>Chapter 4.1.5 --- Objectives --- p.116<br>Chapter 4.2 --- Materials and Methods --- p.118<br>Chapter 4.2.1 --- Isolation of AP --- p.118<br>Chapter 4.2.2 --- Characterization of AP extract --- p.118<br>Chapter 4.2.3 --- Effect of AP on CETP activity in vitro --- p.118<br>Chapter 4.2.4 --- Effect of AP on blood cholesterol in hamsters --- p.119<br>Chapter 4.2.4.1 --- Animals --- p.119<br>Chapter 4.2.4.2 --- Diets --- p.120<br>Chapter 4.2.4.3 --- Plasma lipids measurement --- p.121<br>Chapter 4.2.4.4 --- Plasma CETP activity measurement and immunoreactive mass by Western blotting --- p.123<br>Chapter 4.2.4.5 --- "Measurement of liver SREBP-2, LDL-R, HMG-R and CYP7A1 protein abundance by Western blotting" --- p.124<br>Chapter 4.2.4.6 --- Statistics --- p.124<br>Chapter 4.3 --- Results --- p.125<br>Chapter 4.3.1 --- Polyphenol content in AP --- p.125<br>Chapter 4.3.2 --- Effect of AP on CETP activity in vitro --- p.125<br>Chapter 4.3.3 --- Growth and food intake --- p.128<br>Chapter 4.3.4 --- Organ weights --- p.128<br>Chapter 4.3.5 --- Effect of AP supplementation on the plasma lipid profile of hamsters --- p.131<br>Chapter 4.3.6 --- Effect of AP feeding on plasma CETP activity of the hamsters --- p.131<br>Chapter 4.3.7 --- Effect of AP on plasma CETP immunoreactive mass --- p.134<br>Chapter 4.3.8 --- Effect of AP on liver SREBP-2 immunoreactive mass --- p.134<br>Chapter 4.3.9 --- Effect of AP on liver LDLR immunoreactive mass --- p.134<br>Chapter 4.3.10 --- Effect of AP on liver HMGR immunoreactive mass --- p.134<br>Chapter 4.3.11 --- Effect of AP on liver CYP7A1 immunoreactive mass --- p.134<br>Chapter 4.3.12 --- Effect of AP on liver cholesterol level --- p.140<br>Chapter 4.4 --- Discussion --- p.142<br>Chapter CHAPTER 5 --- CONCLUSION --- p.145<br>REFERENCES --- p.148

Ma, Ka Ying.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.<br>Includes bibliographical references (leaves 113-129).<br>Abstracts in English and Chinese.<br>ACKNOWLEDGMENTS --- p.I<br>ABSTRACT --- p.II<br>LIST OF ABBREVIATIONS --- p.VII<br>TABLE OF CONTENTS --- p.IX<br>Chapter Chapter 1 --- General Introduction --- p.1<br>Chapter 1.1 --- Calcium --- p.1<br>Chapter 1.1.1 --- Recommendation of calcium intake --- p.1<br>Chapter 1.1.2 --- Calcium toxicity --- p.2<br>Chapter 1.1.3 --- Calcium homeostasis --- p.2<br>Chapter 1.1.3.1 --- Role of parathyroid hormone in calcium homeostasis --- p.4<br>Chapter 1.1.3.2 --- "Role of 1,25-dihydroxyvitamin D3 in calcium homeostasis" --- p.4<br>Chapter 1.1.3.3 --- Role of calcitonin in calcium homeostasis --- p.6<br>Chapter 1.2 --- Magnesium --- p.7<br>Chapter 1.2.1 --- Recommendation of magnesium intake --- p.7<br>Chapter 1.2.2 --- Absorption and secretion of magnesium --- p.8<br>Chapter 1.3 --- Cholesterol --- p.9<br>Chapter 1.3.1 --- Cholesterol homeostasis --- p.11<br>Chapter 1.3.1.1 --- Role of LDLR --- p.14<br>Chapter 1.3.1.2 --- Role of SREBP-2 --- p.17<br>Chapter 1.3.1.3 --- HMGR as rate limiting step for cholesterol synthesis --- p.19<br>Chapter 1.3.1.4 --- CYP7A1 as a key factor in production of bile acids --- p.21<br>Chapter 1.3.1.5 --- Role of LXR in production of bile acids --- p.22<br>Chapter 1.3.1.6 --- AC AT regulates cholesterol uptake in intestine --- p.22<br>Chapter Chapter 2 --- Effect of Calcium Deficiency and Inadequacy on Blood Cholesterol Level in Intact Male and Castrated Hamsters --- p.25<br>Chapter 2.1 --- Introduction --- p.25<br>Chapter 2.2 --- Objective --- p.28<br>Chapter 2.3 --- Materials and methods --- p.29<br>Chapter 2.3.1 --- Hamsters --- p.29<br>Chapter 2.3.1.1 --- Intact male hamster --- p.29<br>Chapter 2.3.1.2 --- Castrated hamster --- p.30<br>Chapter 2.3.2 --- Diets --- p.31<br>Chapter 2.3.3 --- Determination of calcium content in animal diet --- p.33<br>Chapter 2.3.4 --- "Determination of serum lipid, lipoproteins and calcium concentration" --- p.33<br>Chapter 2.3.5 --- Determination of cholesterol concentration in organs --- p.34<br>Chapter 2.3.6 --- Determination of fecal neutral and acidic sterols --- p.37<br>Chapter 2.3.7 --- Determination of fecal neutral sterols --- p.37<br>Chapter 2.3.8 --- Determination of fecal acidic sterols --- p.40<br>Chapter 2.3.9 --- Statistics --- p.42<br>Chapter 2.4 --- Results on intact male hamsters --- p.43<br>Chapter 2.4.1 --- Diet composition --- p.43<br>Chapter 2.4.2 --- Growth and food intake --- p.43<br>Chapter 2.4.3 --- Organ weights --- p.43<br>Chapter 2.4.4 --- Effect of calcium deficiency diet on the plasma lipid profile and calcium concentration of hamsters --- p.43<br>Chapter 2.4.5 --- Effect of calcium deficiency diet on hepatic cholesterol of hamsters --- p.44<br>Chapter 2.4.6 --- Effect of calcium on fecal neutral sterol output --- p.48<br>Chapter 2.4.7 --- Effect of calcium on fecal acidic sterol output --- p.48<br>Chapter 2.5 --- Results on castrated hamsters --- p.50<br>Chapter 2.5.1 --- Growth and food intake --- p.50<br>Chapter 2.5.2 --- Organ weights --- p.50<br>Chapter 2.5.3 --- Effect of calcium deficiency diet on the plasma lipid profile and calcium concentration of hamsters --- p.50<br>Chapter 2.5.4 --- Hepatic cholesterol --- p.50<br>Chapter 2.5.5 --- Effect of calcium on fecal neutral sterol output --- p.53<br>Chapter 2.5.6 --- Effect of calcium on fecal acidic sterol output --- p.53<br>Chapter 2.6 --- Discussion --- p.55<br>Chapter Chapter 3 --- Effect of Calcium Deficiency and Inadequacy on Blood Cholesterol Level in Intact Female and Ovariectomized Hamsters --- p.57<br>Chapter 3.1 --- Introduction --- p.57<br>Chapter 3.2 --- Objective --- p.58<br>Chapter 3.3 --- Materials and methods --- p.59<br>Chapter 3.3.1 --- Hamsters --- p.59<br>Chapter 3.3.1.1 --- Intact female hamster --- p.59<br>Chapter 3.3.1.2 --- Ovariectomized hamster --- p.60<br>Chapter 3.3.2 --- Diets --- p.60<br>Chapter 3.3.3 --- "Determination of serum lipid, lipoproteins and calcium concentration" --- p.60<br>Chapter 3.3.4 --- "Determination of cholesterol concentration in organs, fecal neutral and acidic sterols" --- p.60<br>Chapter 3.3.5 --- "Western blottting of liver SREBP-2, LDLR, HMGR, LXR and CYP7A1 proteins" --- p.61<br>Chapter 3.3.6 --- Preparation of intestinal microsome --- p.62<br>Chapter 3.3.7 --- Intestinal acyl coenzyme A: cholesterol acyltransferase (ACAT) activity measurement --- p.63<br>Chapter 3.3.8 --- Statistics --- p.64<br>Chapter 3.4 --- Results on intact female hamsters --- p.65<br>Chapter 3.4.1 --- Growth and food intake --- p.65<br>Chapter 3.4.2 --- Organ weights --- p.65<br>Chapter 3.4.3 --- Effect of calcium deficiency diet on the plasma lipid profile and calcium concentration of hamsters --- p.65<br>Chapter 3.4.4 --- Effect of calcium deficiency diet on hepatic cholesterol of hamsters --- p.65<br>Chapter 3.4.5 --- Effect of dietary calcium on fecal neutral sterol output --- p.66<br>Chapter 3.4.6 --- Effect of dietary calcium on fecal acidic sterol output --- p.66<br>Chapter 3.4.7 --- Effect of dietary calcium on liver LDLR immunoreactive mass --- p.71<br>Chapter 3.4.8 --- Effect of dietary calcium on liver CYP7A1 immunoreactive mass --- p.71<br>Chapter 3.4.9 --- Effect of dietary calcium on liver LXR immunoreactive mass --- p.71<br>Chapter 3.4.10 --- Effect of dietary calcium on liver SREBP-2 immunoreactive mass --- p.71<br>Chapter 3.4.11 --- Effect of dietary calcium on liver HMGR immunoreactive mass --- p.71<br>Chapter 3.4.12 --- Effect of dietary calcium deficiency on intestinal ACAT activity --- p.77<br>Chapter 3.5 --- Results on ovariectomized hamsters --- p.79<br>Chapter 3.5.1 --- Growth and food intake --- p.79<br>Chapter 3.5.2 --- Organ weights --- p.79<br>Chapter 3.5.3 --- Effect of calcium deficiency diet on plasma lipid profile and calcium concentration of hamsters --- p.79<br>Chapter 3.5.4 --- Hepatic cholesterol --- p.79<br>Chapter 3.5.5 --- Effect of dietary calcium on fecal neutral sterol output --- p.80<br>Chapter 3.5.6 --- Effect of dietary calcium on fecal acidic sterol output --- p.80<br>Chapter 3.5.7 --- Effect of dietary calcium on liver LDLR immunoreactive mass --- p.85<br>Chapter 3.5.8 --- Effect of dietary calcium on liver CYP7A1 immunoreactive mass --- p.85<br>Chapter 3.5.9 --- Effect of dietary calcium on liver LXR immunoreactive mass --- p.85<br>Chapter 3.5.10 --- Effect of dietary calcium on liver SREBP-2 immunoreactive mass --- p.85<br>Chapter 3.5.11 --- Effect of dietary calcium on liver HMGR immunoreactive mass … --- p.85<br>Chapter 3.6 --- Discussion --- p.91<br>Chapter Chapter 4 --- Effect of Dietary Magnesium Supplementation on Blood Cholesterol Level in Intact Male Hamsters --- p.94<br>Chapter 4.1 --- Introduction --- p.94<br>Chapter 4.2 --- Objective --- p.96<br>Chapter 4.3 --- Materials and methods --- p.97<br>Chapter 4.3.1 --- Hamsters --- p.97<br>Chapter 4.3.2 --- Diets --- p.98<br>Chapter 4.3.3 --- "Determination of serum lipid, lipoproteins and magnesium concentration" --- p.100<br>Chapter 4.3.4 --- "Determination of cholesterol concentration in organ, fecal neutral and acidic sterols" --- p.100<br>Chapter 4.3.5 --- Statistics --- p.100<br>Chapter 4.4 --- Results on male hamster --- p.101<br>Chapter 4.4.1 --- Growth and food intake --- p.101<br>Chapter 4.4.2 --- Organ weights --- p.101<br>Chapter 4.4.3 --- Effect of dietary magnesium on plasma lipid profile and magnesium concentration in hamsters --- p.101<br>Chapter 4.4.4 --- Effect of dietary magnesium on hepatic cholesterol of hamsters..… --- p.102<br>Chapter 4.4.5 --- Effect of dietary magnesium on fecal neutral sterol output --- p.105<br>Chapter 4.4.6 --- Effect of dietary magnesium on fecal acidic sterol output --- p.105<br>Chapter 4.6 --- Discussion --- p.107<br>Chapter Chapter 5 --- Conclusion --- p.110<br>References --- p.113

非傳染性疾病是目前全球最常見的疾病之一。不健康的食相信是導致非傳染性疾病增加的主要因素之一。因此，我們就食對乳腺癌的形成和膽固醇代謝調控的影響進行了研究。<br>在去除卵巢的祼鼠模型中，我們研究了長期和短期熱量限制對乳腺癌腫瘤增殖的影響。14週齡的小鼠被隨機分為5組：自由攝食組 (AL)；熱量攝入控制在AL80% 的20%CCR組；熱量攝入控制在AL的70% 的30%CCR組；熱量攝入控制在AL的65% 的35%CCR組和短期熱量限制 （SCR）組 (前3.5週熱量攝入控制在AL的65%，之後的13.5週自由攝食)。10週後，熱量限制組的腫瘤體積明顯較AL組小 (P < 0.05)。排除攝食對體重的影響，SCR組的腫瘤重量明顯較AL組小 (P < 0.05)。本實驗結果表明，在此動物模型中，短期熱量限制能有效抑制乳腺癌細胞的增殖。<br>此外，我們還研究了芹菜素在肝細胞中對膽固醇代謝的影響。芹菜素是一種常見的黃酮類化合物。研究發現，在WRL-68細胞中，芹菜素能夠劑量依賴性的抑制3 - 羥基-3 - 甲基 - 戊二酸單酰輔酶還原酶 (HMGCR)和固醇調節元件結合蛋白-2 (SREBP-2) 信使RNA和蛋白的表達及其啟動子的轉錄活性。綜上所述，在肝細胞中，芹菜素能有效抑制HMGCR和SREBP-2的表達，從而達到降低膽固醇的效果。<br>總括而言，本研究表明在去除卵巢的祼鼠模型中，短期熱量限制能有效抑制乳腺癌細胞的生長和芹菜素能有效抑制HMGCR和SREBP-2的表達。<br>Non-communicable diseases (NCD) are one of the leading causes of mortality in the developed and under-developing countries. Diet is a major risk factor of NCD. In the present study, effects of diet modification on breast cancer development and cholesterol metabolism were investigated.<br>In the first part of this study, the effect of chronic and short-term calorie restriction (CR) on breast tumor growth in ovariectomized nude mice was investigated. The calorie-restricted dietary regimen limited the total fat intake only. 14 week-old ovariectomized female nude mice were randomly assigned to ad libitum fed (AL), 20%CCR (17-week 80% of AL), 30%CCR (17-week 70% of AL), 35%CCR (17-week 65% of AL) and short-term CR (3.5-week 65% of AL followed by 13.5-week 100% AL consumption) groups. Starting from 10 weeks after transplant of cells, the tumor volumes in all calorie-restricted groups were significantly smaller (P < 0.05) than that in ad libitum control. At sacrifice, the tumor weight in short-term CR was significantly smaller (P < 0.05) than that in ad-libitum control after normalized with body weight. This indicated that short-term CR could suppress tumor in this model.<br>In the second part of this study, the effect of apigenin on cholesterol metabolism was investigated. Apigenin is one of the most abundant flavonoids. In the present study, we investigated the effect of apigenin on several cholesterol-related gene expression in hepatic cells. In WRL-68 cells treated with apigenin, promoter transcription activity, mRNA and protein expression of HMGCR and SREBP-2 were significantly decreased in a dose-dependent manner. Taken together, we concluded that apigenin inhibited HMGCR and SREBP-2 gene expressions in hepatic cells, which might elicit the hypocholesterolemic effects.<br>In conclusion, our study has demonstrated that short-term CR could significantly block the breast tumor growth in a mice model and apigenin could inhibit the expression of HMGCR and SREBP-2 in liver cell lines.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Wong, Tsz Yan.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 83-99).<br>Abstracts also in Chinese.<br>ACKNOWLEGEMENTS --- p.i<br>ABSTRACT --- p.ii<br>摘要 --- p.iv<br>list of abbreviations --- p.v<br>list of figures --- p.vii<br>list of tables --- p.IX<br>TABLE of CONTENTS --- p.X<br>Chapter 1 --- CHAPTER 1 --- p.1<br>General Introduction --- p.1<br>Chapter 1.1 --- Calorie Restriction and the Prevention of Postmenopausal Breast Cancer --- p.2<br>Chapter 1.1.1 --- Breast Cancer --- p.2<br>Chapter 1.1.2 --- Epidemiology of Excess Body Weight and Cancer Risk --- p.3<br>Chapter 1.1.3 --- Calorie Restriction and Cancer Prevention --- p.7<br>Chapter 1.1.4 --- Mechanistic Targets of Calorie Restriction --- p.8<br>Chapter 1.1.4.1 --- Effect of Calorie Restriction on Estrogen --- p.8<br>Chapter 1.1.4.1 --- Effect of Calorie Restriction on Cell Cycle Regulation --- p.12<br>Chapter 1.1.4.1 --- Effect of Calorie Restriction on Apoptosis --- p.14<br>Chapter 1.2 --- Effect of Apigenin on Cholesterol Homeostasis --- p.17<br>Chapter 1.2.1 --- Cardiovascular Disease and Blood Cholesterol --- p.17<br>Chapter 1.2.2 --- Molecular Regulation of Cholesterol Metabolism --- p.21<br>Chapter 1.2.2.1 --- HMG-CoA Reductase --- p.21<br>Chapter 1.2.2.2 --- CYP7A1 --- p.24<br>Chapter 1.2.2.3 --- Apolipoprotein A-1 --- p.26<br>Chapter 1.2.2.4 --- Low Density Lipoprotein Receptor --- p.29<br>Chapter 1.2.2.5 --- Sterol Regulatory Element Binding Proteins --- p.31<br>Chapter 1.2.3 --- Flavonoid and its Association with Cholesterol Metabolism --- p.36<br>Chapter 1.2.4 --- Apigenin: A Potential Alternative --- p.39<br>Chapter 2 --- CHAPTER 2 --- p.41<br>MATERIALS AND METHODS --- p.41<br>Chapter 2.1 --- Chemicals and Materials --- p.41<br>Chapter 2.1.1 --- Chemicals --- p.41<br>Chapter 2.1.2 --- Plasmids --- p.41<br>Chapter 2.2 --- Cell Culture --- p.41<br>Chapter 2.2.1 --- Maintainance of Cells --- p.41<br>Chapter 2.2.2 --- Preparation of Cell Stock --- p.42<br>Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.42<br>Chapter 2.3 --- Measurement of Cell viability --- p.43<br>Chapter 2.4 --- Semi-Quantitative and Quantitative RT-PCR Assay --- p.43<br>Chapter 2.4.1 --- RNA Isolation and cDNA Synthesis --- p.43<br>Chapter 2.4.2 --- Quantitative Real Time PCR Assay --- p.43<br>Chapter 2.4.2.1 --- Real Time PCR Using TaqMan Probe --- p.43<br>Chapter 2.4.2.2 --- Real Time PCR Using SYBR Green Dye --- p.44<br>Chapter 2.4.2.3 --- Statistical Analysis of 2⁻ΔΔ{U+A7F0}{U+1D40} Comparative Gene Expression --- p.44<br>Chapter 2.5 --- Western Blot Analysis --- p.46<br>Chapter 2.6 --- Measurement of Promoter Activity --- p.46<br>Chapter 2.6.1 --- Plasmid Preparation --- p.46<br>Chapter 2.6.2 --- Transient Transfection and Dual-Luciferase Assay --- p.47<br>Chapter 2.7 --- Animal Experiment Design --- p.47<br>Chapter 2.7.1 --- Animal Model and Dietary Regimens --- p.47<br>Chapter 2.7.2 --- Tissue Sample Collection --- p.50<br>Chapter 2.7.3 --- Plasma Estradiol Determination --- p.50<br>Chapter 2.7.4 --- Protein and RNA extraction --- p.50<br>Chapter 2.8 --- Statistical Analysis --- p.50<br>Chapter 3 --- Chapter 3 --- p.51<br>EFFECT OF CHRONIC AND short-term calorie restriction on breast tumor growth in ovariectomized nude mice --- p.51<br>Chapter 3.1 --- Introduction --- p.51<br>Chapter 3.2 --- Objectives --- p.52<br>Chapter 3.3 --- Results --- p.53<br>Chapter 3.3.1 --- Food Intakes, Body, Liver and Uterus Wet Weights of the Mice --- p.53<br>Chapter 3.3.2 --- Tumor Development --- p.57<br>Chapter 3.3.3 --- Plasma Estradiol Level --- p.62<br>Chapter 3.3.4 --- Estradiol Responsive Gene expression in Tumors --- p.63<br>Chapter 3.3.5 --- Cell Apoptotic and Cell Cycle-Regulated Protein expression in Tumors --- p.65<br>Chapter 3.4 --- Discussion --- p.67<br>Chapter 4 --- CHAPTER 4 --- p.69<br>Apigenin inhibits the expression of hmg-coa reductase and srebp-2 in hepatic cells --- p.69<br>Chapter 4.1 --- Introduction --- p.69<br>Chapter 4.2 --- Objectives --- p.70<br>Chapter 4.3 --- Results --- p.70<br>Chapter 4.3.1 --- Effect of Apigenin on Cell Viability --- p.70<br>Chapter 4.3.2 --- Effect of Apigenin on HMGCR, CYP7A1, LDLR, ApoA-1, SREBP-1 and SREBP-2 mRNA expressions --- p.72<br>Chapter 4.3.3 --- Effect of Apigenin on HMGCR, LDLR, ApoA-1 and SREBP-2 Promoter Transcription Activity --- p.75<br>Chapter 4.3.4 --- Effect of Apigenin on HMGCR, SREBP-1 and SREBP-2 Protein Expression --- p.77<br>Chapter 4.3.5 --- Role of Estrogen Receptor in Apigenin induced SREBP-2 Inhibition --- p.79<br>Chapter 4.4 --- Discussion --- p.80<br>Chapter 5 --- CHAPTER 5 --- p.82<br>SUMMARY --- p.82<br>References --- p.83

Indiana University-Purdue University Indianapolis (IUPUI)<br>Excess caloric intake and/or obesity currently remain the largest predisposing risk factors for the development of type 2 diabetes. Discerning the cellular and molecular mechanisms responsible and amendable to therapy represents a growing challenge in medicine. At a cellular level, increased activity of the hexosamine biosynthesis pathway (HBP), a sensor of excess energy status, has been suggested to promote the exacerbation of insulin resistance through increasing adipose tissue and skeletal muscle membrane cholesterol content. This in turn compromises cortical filamentous actin structure necessary for proper incorporation of the insulin-sensitive glucose transporter GLUT4 into the plasma membrane. The current studies attempted to elucidate the mechanism by which hexosamines provoke membrane cholesterol toxicity and insulin resistance. In 3T3-L1 adipocytes cultured with pathophysiologic hyperinsulinemia to induce insulin resistance, increased HBP flux was observed. This occurred concomitant with gains in the mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Mechanistically, immunoprecipitation demonstrated increased HBP-induced N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), a regulator of HMGR synthesis. This was associated with increased affinity toward and activity of Hmgcr, the gene encoding HMGR. Global HBP inhibition or Sp1 binding to DNA prevented membrane cholesterol accrual, filamentous actin loss, and glucose transport dysfunction. Furthermore, hyperinsulinemia and HBP activation impaired cholesterol efflux in adipocytes, exacerbating cholesterol toxicity and potentially contributing to cardiovascular disease. In this regard, chromium picolinate (CrPic), known to have beneficial effects on glucose and lipoprotein metabolism, improved cholesterol efflux and restored membrane cholesterol content. To test the role of membrane cholesterol accumulation in vivo, studies were conducted on C57Bl/6J mice fed a low or high fat diet. High fat feeding promoted increased HBP activity, membrane cholesterol accumulation, and insulin resistance. Supplementation of mice with CrPic in their drinking water (8µg/kg/day) countered these derangements and improved insulin sensitivity. Together, these data provide mechanistic insight for the role of membrane cholesterol stress in the development of insulin resistance, as well as cardiovascular disease, and highlight a novel therapeutic action of chromium entailing inhibition of the HBP pathway.

碩士<br>國立臺灣海洋大學<br>食品科學系<br>92<br>This research was intended to investigate the effects of fermented soymilk containing N-acetylchitooligosaccharides on intestinal physiology and immuno-activity of male Wistar rats fed a high fat and a high cholesterol diet (6% corn oil or 10% lard and 0.5% cholesterol). The immuno-activity of fermented product also checked by in vitro experiments. The diets for rats used in the study were divided into four groups: (1) soymilk containing N-acetylchitooligosaccharides (oligomer soymilk), (2) fermented soymilk containing N-acetylchitooligosaccharides (oligomer fermented soymilk), (3) fermented soymilk, (4) soymilk (control). A 10% (v/v) chitin hydrolysate was added into soymilk according the design purposes. Lactobacillus plantarum BCRC 12250 and Streptococcus thermophilus BCRC 12268 were used as the starters of fermented soymilk. The fermentation was conducted at 37℃ for three days until the pH of the product revealed 4.6. The soymilk powder was added into the experimental diets after drying by spray-dry. The experiment was lasted for four weeks. During feeding, the ad libitum was carried out. The dried sample powder was used to co-incubate with cell line in in vitro experiment to assess the effects to cells. Results showed that the total cholesterol and total triglyceride in plasma of rats fed the oligomer fermented soymilk were lower than those of control. There was no difference at GOT, GPT, and LDH in plasma among groups. The spleen lymphocytes proliferation and the phagocytosis of peritoneal exudates cells of rats fed on oligomer fermented soymilk were more than that of control group. In in vitro test, the oligomer fermented soymilk could not only enhance the proliferation of cell line UMΦ and HB4C5, but also stimulate the mice spleen lymphocytes to secrete more IgG and IgM.

Indiana University-Purdue University Indianapolis (IUPUI)<br>A recent review of randomized controlled trials found that trivalent chromium (Cr3+) supplementation significantly improved glycemia among patients with diabetes, consistent with a long-standing appreciation that this micronutrient optimizes carbohydrate metabolism. Nevertheless, a clear limitation in the current evidence is a lack of understanding of Cr3+ action. We tested if increased AMP-activated protein kinase (AMPK) activity, previously observed in Cr3+-treated cells or tissues from Cr3+-supplemented animals, mediates improved glucose transport regulation under insulin-resistant hyperinsulinemic conditions. In L6 myotubes stably expressing the glucose transporter GLUT4 carrying an exofacial myc-epitope tag, acute insulin stimulation increased GLUT4myc translocation by 69% and glucose uptake by 97%. In contrast, the hyperinsulinemic state impaired insulin stimulation of these processes. Consistent with Cr3+’s beneficial effect on glycemic status, chromium picolinate (CrPic) restored insulin’s ability to fully regulate GLUT4myc translocation and glucose transport. Insulin-resistant myotubes did not display impaired insulin signaling, nor did CrPic amplify insulin signaling. However, CrPic normalized elevated membrane cholesterol that impaired cortical filamentous actin (F-actin) structure. Mechanistically, data support that CrPic lowered membrane cholesterol via AMPK. Consistent with this data, siRNA-mediated AMPK silencing blocked CrPic’s beneficial effects on GLUT4 and glucose transport regulation. Furthermore, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) protected against hyperinsulinemia-induced membrane/cytoskeletal defects and GLUT4 dysregulation. To next test Cr3+ action in vivo, we utilized obesity-prone C57Bl/6J mice fed a low fat (LF) or high fat (HF) diet for eight weeks without or with CrPic supplementation administered in the drinking water (8 µg/kg/day). HF feeding increased body weight beginning four weeks after diet intervention regardless of CrPic supplementation and was independent of changes in food consumption. Early CrPic supplementation during a five week acclimation period protected against glucose intolerance induced by the subsequent eight weeks of HF feeding. As observed in other insulin-resistant animal models, skeletal muscle from HF-fed mice displayed membrane cholesterol accrual and loss of F-actin. Skeletal muscle from CrPic-supplemented HF-fed mice showed increased AMPK activity and protection against membrane cholesterol accrual and F-actin loss. Together these data suggest a mechanism by which Cr3+ may positively impact glycemic status, thereby stressing a plausible beneficial action of Cr3+ in glucose homeostasis.