Thesis (Ph.D.)--East Carolina University, 2009. / Presented to the faculty of the Department of Exercise and Sports Science. Advisor: Joseph A. Houmard. Title from PDF t.p. (viewed Apr. 30, 2010). Includes bibliographical references.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Risks of metabolic syndrome including cardiovascular disease and diabetes are significantly affected by maternal nutrition. This concept of developmental programming had been investigated in our laboratory and in an earlier study, it was established that maternal high-fat diet predisposed rat offspring to insulin resistance and higher triglyceride in serum, liver, skeletal muscle and adipose tissue. These abnormalities, however, were ameliorated by supplementing green tea extract to dam’s diet throughout gestation and lactation. The overall objective of this thesis was to examine lipid metabolism, oxidative stress and inflammatory responses in heart of offspring born to dams receiving high-fat diet with or without green tea supplementation during pregnancy and lactation. Female Sprague-Dawley rats were fed an obesogenic diet which was a high-fat diet (HF,30%), low-fat diet (LF,7%) or HF diet containing 0.75% green tea extract prior to conception and throughout gestation. During lactation, half of the dams had their diet switched from HF to GT and vice versa. Pups were weaned to the HF or LF diet, forming 10 offspring groups (gestation/lactation/postweaning): LF/LF/LF, LF/LF/HF, HF/HF/LF, HF/HF/HF, HF/GT/LF, HF/GT/HF, GT/GT/LF, GT/GT/HF, GT/HF/LF and GT/HF/HF. Except a larger fibrotic area, maternal HF diet did not affect lipid accumulation, oxidative status and inflammatory response in the heart of offspring. Analysis of variance revealed different, and even opposite, effects of GT supplementation during gestation and lactation. In offspring born to dams receiving GT supplementation during gestation, they had suppressed fatty acid oxidation (FAO) and higher triglyceride (TG) level in the heart. In contrast, when GT was supplemented to dams during lactation, offspring had elevated heart TG, cholesterol and free fatty acid levels but up-regulated FAO. Since FAO is associated with reactive oxygen species (ROS) production, modulation of FAO is believed to affect cellular stress responses in heart. Consistent with FAO, cardiac stress, apoptotic and inflammatory biomarkers including B-type natriuretic peptide (BNP), bcl 2 associated-x (bax) and interleukin-1β (IL1b) were down-regulated in offspring born to dams given GT during gestation, whereas GT supplementation during lactation increased the expression of pro-apoptotic markers: bax and caspase-3 (Cas3) concurrent with activation of antioxidant defense system: catalase, glutathione peroxidase (GPx) and glutathione S-transferase (GST) as adaptive mechanism against increased ROS. Uncoupling protein 2 (UCP 2) and subsequent higher bcl 2 /bax ratio has been reported to stimulate apoptosis. In agreement with this, mRNA expression of BNP, bax and Cas3 were found to correlate with that of UCP 2. This suggests UCP 2may play an important role in apoptosis under the impact of maternal GT supplementation. The present data suggest that the effect of maternal high-fat diet is organ specific causing apparently lesser damage to the heart. When GT is given in conjunction with a high-fat diet to dams during gestation, there is no clear cut advantage to the offspring. However, potential adverse effects could not be ruled out when GT is supplemented to dams during lactation possibly due to higher catechin exposure via milk. Future study should focus on establishing the benefits and safety use of GT during gestation. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Introduction: The relationship between pancreatitis and dyslipidaemia is unclear and has never been studied in a South African context. Patients and methods: A prospective evaluation of all admissions with acute pancreatitis to a regional hospital general surgical service was performed to ascertain its relationship to dyslipidaemia. Aetiology was determined by history and ultrasound assessment. Disease severity was assessed using a modified Imrie score and an organ failure score. Body mass index was calculated. A lipid profile was obtained. Abnormal profiles were repeated. Secondary causes of dyslipidaemia were noted. A comparison of the demographic profile, aetiology, disease severity scores, complications and deaths were made in relationship to the lipid profiles. Results: From June 2001 to May 2005, there were 230 admissions, of whom 31% were women and 69% men. The median age was 38 years(range 13- 73). The pancreatitis was associated with alcohol in 146(63%), gallstones in 42(19%) and idiopathic in 27(12%). The amylase was significantly higher with a gallstone aetiology (p / Thesis (MMedSc)-University of KwaZulu-Natal, 2006.

To investigate interactive effects of dietary fatty acid composition and energy restriction on body lipid metabolism and its regulation, rats were fed for 10 weeks diets varying in fat type and energy intake level. Energy deficiency was achieved by removing carbohydrate from the diets while keeping fat and other nutrient intakes constant. Tissue fatty acid deposition was influenced by the interaction between the dietary fat source and body energy balance. Less total fatty acids were deposited in livers of the ad libitum beef tallow-fed animals than the other fat feedings. However, such difference no longer existed when energy intake was restricted. Similarly, less energy supply eliminated the higher docosahexaenoic acid and lower arachidonic acid contents associated with the fish oil feeding in hepatocyte membrane phosphatidylchohne, phosphatidylserine and sphingomyelin. Tissue lipogenesis was also examined as a function of the interaction of dietary fatty acid composition and energy restriction. Comparable absolute cholesterol synthesis rates were observed in livers of the food restricted animals fed different types of dietary fat, although the synthesis rates were different among the dietary fat groups fed ad libitum. Energy restriction increased the triglyceride-fatty acid synthesis rates in the intestine of the fish and safflower oil-fed groups, but not in that of the olive oil- and beef tallow-fed animals. Plasma leptin concentrations were 60% higher in the ad libitum-fed fish and safflower oil groups as compared with those in the beef tallow diet group, despite smaller perirenal fat mass and fat cell size in the fish oil-fed animals. Energy restriction decreased plasma leptin levels of the fish and safflower oil-fed rats, but not those in the beef tallow-fed animals. Taken together, these results demonstrate that the structural, functional and regulating aspects of tissue lipid metabolism were influenced by an interaction between dietary fatty acid composit

Japanese quail hens were used to study the effect of feeding palmitic, oleic or linoleic acids on the reproductive performance, tissue fatty acid composition and embryo lipid metabolism. Quail fed palmitic acid consumed more feed than those fed either oleic or linoleic acids. The highest level of reproductive performance was observed in quail fed palmitic acid followed by those fed oleic and linoleic acids. The highest level of embryo survival, observed in the palmitic acid fed group, was associated with more rapid mobilization and assimilation of yolk material by the embryo during incubation and it was not related to changes in eggshell quality. High levels of oleic and linoleic acids were found in egg yolk, plasma and liver lipids from quail fed oleic and linoleic acids, respectively. However, feeding palmitic acid resulted in elevated levels of palmitoleic acids in all three tissues. The fatty acid profiles of phospholipid, triglyceride and cholesterol esters of embryonic tissues were consistently influenced by the fatty acid composition of the yolk lipids and the stage of development. Feeding palmitic acid promoted more retention of labeled fatty acids in embryo lipids. Labeled oleic acid was preferentially esterified in the cholesterol ester fraction of yolk sac membrane lipids, and it appears that this fatty acid is utilized to a great extent by the quail embryo during its development.

Apolipoprotein C-I (apoC-I) plays an important role in the metabolism of plasma triglyceride levels and cholesterol metabolism. Little is known about the regulation of apoC-I production by human adipocytes. Aim. To investigate the effect of different tissue culture conditions on the synthesis and secretion of apoC-I and apoE in human SW872 liposarcoma cells and to study the effects of apoC-I overexpression in these same cells. Methods. SW872 cells were grown in DMEM/F-12 (3:1, v/v). QPCR was used to quantify mRNA synthesis. ELISAs were used to quantify intracellular and extracellular proteins. Colorimetric reaction kits were used to analyze intracellular cholesterol and triglyceride concentrations. Results . Maturation experiments revealed that after 17 days in culture, SW872 cells contained significantly more cholesterol (100%) and triglyceride (3-fold). Cell maturation was associated with significantly higher levels of apoE mRNA (+200%) but not apoC-I mRNA (-50%). The cells secreted more apoC-I (+110%) and apoE (+340%). Cellular apoC-I increased 620% and apoE increased 1540%. Treatment of cells during maturation with insulin (0, 10 or 1000 nM) significantly reduced the secretion of apoC-I and apoE (-14% and -56%, respectively) and intracellular apoC-I and apoE (-10% and -12%, respectively. Overexpression of apoC-I in SW872 cells resulted in increased cell number (+70%) and decreased lipids per cell (-32% triglyceride, -36% cholesterol) as compared to controls. Conclusion. These results suggest that apoC-I and apoE production is differentially regulated at the transcriptional level in adipocytes and that apoC-I and apoE play a role in the maturation of human adipocytes and may have an important role in mediating or regulating cell lipid accumulation. As well, overexpression of apoC-I in SW872 cells impedes cellular lipid accumulation and stimulates cellular proliferation.

No description available.

No description available.

A nonsynonymous, loss of function variant (rs58542926, E167K) located in the gene encoding TM6SF2 was identified in multiple genetic association studies as significantly correlating with increased risk for non-alcoholic fatty liver disease (NAFLD) and decreased risk for hyperlipidemia. Given the pivotal role that lipoproteins play at the juncture of these two conditions, researchers hypothesize that the ER-membrane spanning TM6SF2 protein regulates the degree of lipidation of VLDL particles synthesized in the liver. However, not all published data supports this theory and contradictions regarding many aspects of the mechanistic function of TM6SF2 remain. The inconsistencies observed in the literature are in part due to drawbacks of the models used to study TM6SF2 activity; thus, there is an obvious need for an improved hepatocyte model to better understand how TM6SF2 impacts lipid metabolism.To address this need, we present an optimized protocol for the differentiation of inducible pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs), created in collaboration with the Leong Lab. We provide extensive validation of HLC maturity and hepatic functionality, including prolonged albumin secretion, evidence of membrane polarity, and cytochrome P450 induction. We also demonstrate that HLCs express proteins essential for lipoprotein metabolism, secrete authentic VLDL particles, and respond to metabolic perturbations, supporting their value for modeling hepatosteatosis and VLDL metabolism in vitro. To investigate the effect of TM6SF2 variant expression on hepatic lipid metabolism, we produced HLCs derived from 4 homozygous TM6SF2-carrier individuals (KK) and 4 age- and sex-matched unaffected siblings (EE). We describe the variability in differentiation efficiency that we observed in our sibling-matched HLC model and present the gene editing strategy we developed using CRISPR/Cas9 technology and transgene-induced expression to create isogenic iPSCs differing only in their TM6SF2 genotype [EE, KK, or knockout (KO)]. After extensive confirmation of successful gene editing, we explore the effect of TM6SF2 on lipid metabolism in the edited iPSCs. RNA-sequencing and qPCR validation reveal that the Sterol Regulatory Element Binding Protein 2 (SREBP2)-mediated transcriptional program regulating cholesterol synthesis is significantly increased in TM6SF2 KO iPSCs. However, lipidomics analysis and de novo lipogenesis functional assays show that free cholesterol (FC) levels are unchanged. In TM6SF2 KO iPSCs, we further show a reduction in the activities of Acyl-Coenzyme A: Cholesterol Acyltransferase 1 (ACAT1) and Phosphatidylserine Synthase 1 (PSS1), two enzymes that display optimal function when specifically localized to cholesterol enriched ER lipid raft-like domains. Our findings suggest that TM6SF2 may impact cholesterol localization within ER subdomains, which regulate expression levels of cholesterol synthesis genes and activities of ER lipid-raft associated enzymes. In summary, we present here methodological approaches for generating multiple cell culture models in which to investigate the function of TM6SF2, as well as novel evidence supporting a role for TM6SF2 in iPSC cholesterol metabolism.