The chicken has long been a model used for the study of plasma lipoproteins due to the ability to increase VLDL production by administration of estrogen. In this study we were able to demonstrate successful isolation of VLDL assembly intermediates from the livers of hens, roosters, and estrogen treated rosters. Particle diameter of first step particles, as determined by dynamic laser light scattering, was decreased from an average diameter of 31.5 nm in untreated birds, to 16.1 nm 12 hours after estrogen treatment. Effects of estrogen waned after 24 hours and particle diameter of first step particles increased to an average of 23.9 nm. These assembly intermediates, as well as plasma VLDL and VLDLy, were successfully studied using capillary electrophoresis (CE). Effective mobilities of intact plasma VLDL and first step particles decreased after estrogen administration. Hen VLDL showed a single uniform peak whereas rooster VLDL separated into distinct “subclasses”. Delipidated VLDL, VLDLy and first step assembly intermediates were also successfully separated using CE. This thesis is dedicated to my family who always encouraged me through this process.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 3 uppsatser.

Toxicity preventing activity (TxPA) is a recently identified substance in serum which counteracts the toxic effect of very low density lipoproteins upon endothelial cells in vitro. In two clinical studies, TxPA was low in individuals with angiographically demonstrable coronary artery disease. An atherogenic index which combines TxPA with lipoprotein cholesterol values classifies individuals with coronary artery disease with an accuracy of greater than 93%. TxPA precipitates with 0.15 M trichloroacetic acid and above 3 M (NH4)2SO4. Activity is present in Cohn fractions IV4 and V and is stabilized by antioxidants. TxPA co-lutes with the albumin peak on gel filtration chromatography and as a subcomponent of albumin on ion-exchange chromatography. Isoelectric focusing resolves albumin into two major peaks with pI values of 4.8 and 5.6. The TxPA is identified as the pI 5.6 albumin peak.

This report demonstrates the utilization of a new serum factor, Toxicity Preventing Activity (TxPA) in the diagnosis of coronary disease prone individuals. Our laboratory has recently identified TxPA, which offsets the toxicity of very low density lipoproteins (VLDL) upon arterial cells in vitro. In the present study, we measured TxPA activity and serum lipoprotein levels in 73 individuals undergoing coronary angiography. Serum from control subjects demonstrated 270% more TxPA than aged matched individuals with angiographically demonstrable coronary disease (CHD). When TxPA was combined with serum lipoprotein values, a new atherogenic index was generated which further distinguished these individuals with CHD from non-angiographed controls. These results demonstrate that TxPA is a new protective factor in coronary artery disease, and that the new atherogenic index provides for the first time an accurate classification of individuals with coronary artery disease.

Dietary fats are mainly transported by the intestine in lipoproteins: chylomicrons (CMs) and very low density lipoproteins (VLDLs). Unfortunately, studies of the intestinal absorption of dietary fat have been hampered by the lack of an adequate in vitro model system. As an in vitro model Caco-2 cells are able to secrete lipoproteins. We investigated the possible factors that may affect the secretion of CMs through the ultracentrifugation technique. The dose-dependent effects of oleic acid, mono-olein, egg lecithin, collagen matrix, and the effect of cell differentiation on CM secretion were then tested. We found that oleic acid, lecithin, and cell differentiation are critical for CM secretion by Caco-2 cells. To further confirm that our optimal condition is, in fact, favorable for efficient CM production, we compared it with control groups. We observed that our condition led to more efficient CM secretion as determined by the TGs, ApoB, and TEM analysis.

Cardiovascular diseases (CVDs) have been recorded as the number one cause of death worldwide, accounting for 32% of total deaths annually. More than two-thirds of all CVD cases are associated with atherosclerosis, which is the accumulation of fats and other substances causing plaque formation in the interior walls of major arteries. This leads to narrowing of the lumen and hardening of the arteries, ultimately resulting in angina, heart attack and/or stroke. Studies have shown that the pathogenesis of atherosclerosis and associated CVDs is strongly linked to elevated secretion of liver-specific lipoproteins called very-low-density-lipoprotein (VLDL). VLDLs are crucial lipoproteins responsible for transportation of triacylglycerides (TAGs), chemically inert particles that are physiologically significant for their energy storing capacity, from the liver to peripheral tissues. These VLDL particles are synthesized in the lumen of the endoplasmic reticulum (ER) of hepatocytes, transported from the ER to the cis-Golgi in special transport vesicles called VLDL-transport-vesicles (VTVs) and secreted into plasma through a highly regulated secretory pathway. Previous studies from our laboratory have shown that VTV-mediated ER-to-Golgi VLDL trafficking is the rate-limiting step in overall VLDL secretion from hepatocytes into plasma. In this project, we investigated intracellular VLDL trafficking and VLDL secretion in Ames dwarf (Prop1df, df/df) mice, a mutant mouse model homozygous for a recessive mutation at Prop1 gene locus (Prop1df) having deficiency of growth hormone (GH), thyroid stimulating hormone (TSH) and prolactin (PRL). This model is characteristic of prolonged longevity (~50% longer) and improved insulin sensitivity in comparison to their wild-type (N) counterparts. Ames dwarf (df/df) mice have recently been shown to have highly reduced plasma TAG levels, associating them with reduced susceptibility to atherosclerosis and associated CVDs. The underlying mechanism responsible for reduced VLDL secretion in Ames dwarf mice is yet to be characterized. We hypothesize that VTV-mediated trafficking of VLDL is reduced in Ames dwarf mice because of reduced expression of proteins regulating VLDL and VTV formation. To test our hypothesis, we first performed VTV-budding assay using cellular fractions isolated separately from Ames dwarf (df/df) and wild-type (N) mice livers. Our results show a significant (45%) reduction in VTV-budding process in Ames dwarf (df/df) mice compared to wild-type (N). Next we performed 2-dimensional differential gel electrophoresis (2-DIGE) on VTV and whole cell lysate (WCL) samples in order to examine the differences in protein expression and to have highly specific protein separation. ExPASy database was used to analyze protein spots that allowed us in identifying proteins specifically expressed in each of the mouse groups. Employing western blotting, samples (ER, cytosol, VTV and WCL) from both sets of mice were tested for expression levels of VLDL and VTV associated proteins (ApoB100, Sec22b, CideB, MTP, Apo-A1 and Apo-AIV) with ?-actin as the loading control. Significant differences in expression level of these proteins were observed which strongly suggest that the formation of VTV from ER in male Ames dwarf (df/df) mice is reduced compared to wild-type (N). Overall, we conclude that the differential expression of proteins required for VLDL transport causes reduced VLDL secretion in male Ames dwarf (df/df) mice.

Increased secretion of very low-density lipoprotein (VLDL), a triglyceride-rich lipoprotein, by the liver causes hypertriglyceridemia, which is a major risk factor for the development of atherosclerosis. The rate of VLDL-secretion from the liver is determined by its controlled transport from the endoplasmic reticulum (ER) to the Golgi. The ER-to-Golgi transport of newly synthesized VLDL is a complex multi-step process and is mediated by the VLDL transport vesicle (VTV). Once a nascent VLDL particle is synthesized in the lumen of the ER, it triggers the process of VTV-biogenesis and this process requires coat complex II (COPII) proteins that mediate the formation of classical protein transport vesicles (PTV). Even though, both VTV and PTV bud off the same ER at the same time and require the same COPII proteins, their cargos and sizes are different. The VTV specifically exports VLDL to the Golgi and excludes hepatic secretory proteins such as albumin and the size of the VTV is larger (~ 100 -120 nm) than PTV to accommodate VLDL-sized particles. These observations indicate (i) the existence of a sorting mechanism at the level of the ER; and (ii) the involvement of proteins in addition to COPII components. This doctoral thesis is focused on identification of proteins regulating VLDL sorting into the VTV and involved in the biogenesis of the VTV. In order to identify proteins present exclusively in VTV, we have characterized the proteome of VTV, which suggest CideB (cell death-inducing DFF45-like effector b) and SVIP (small VCP/P97 interacting protein) as candidates, present in VTV but excluded from PTV. We further confirmed the finding by performing co-immunoprecipitation studies and confocal microscopy studies. CideB, a 26-kDa protein was found to interact with apolipoprotein iv B100 (apoB 100), the structural protein of VLDL. Moreover, CideB interacts with two of the COPII components, Sar1 and Sec24. VTV generation was examined after blocking CideB by specific antibodies and by silencing CideB in rat primary hepatocytes. Knockdown of CideB in primary hepatocytes showed significant reduction in VTV generation, however, CideB was concentrated in VTV as compared with the ER suggesting its functional role in the sorting of VLDL into the VTV. SVIP, a small (~ 9-kDa) protein was found to interact with Sar1, a COPII component that initiates the budding of vesicles from ER membrane. SVIP has sites for myristoylation and we found increased recruitment of SVIP on ER membrane upon myristic acid (MA) treatment. Sar1 that lacks sites for myristoylation also is recruited more on ER upon myristoylation indicating that SVIP promotes Sar1 recruitment on ER. Additionally, our data suggest that Sar1 interacts with SVIP and forms a multimer that facilitates the biogenesis of VTV. Interestingly, silencing of SVIP reduced the VTV generation significantly. Conversely, incubation with MA increased the VTV budding, suggesting recruitment of SVIP on ER surface facilitates the VTV budding. We conclude that SVIP recruits Sar1 on ER membrane and makes an intricate COPII coat leading to the formation of a large vesicle, the VTV. Overall, the data presented in this thesis, determines the role of CideB and SVIP in regulating VLDL sorting and VTV biogenesis.

Hypercholesterolemia represents a major risk factor of cardiovascular disease. A number of experimental models is used for study of hypercholesterolemia pathogenesis and therapy. This thesis concentrates on characterization of one of these models. Prague hereditary hypercholesterolemic (PHHC) rat is such a suitable model for study of hypercholesterolemia. Although the majority of plasma cholesterol is transported by high density lipoprotein in PHHC rat fed standard diet, PHHC rat fed cholesterol diet develops hypercholesterolemia comparable to that of humans. The advantage of this model is that hypercholesterolemia develops without the need for addition of bile acids or other hepatotoxic substances to the diet. The hypercholesterolemia of PHHC rat is caused by slowed down catabolism of cholesterol-rich very low density lipoprotein (VLDL). These cholesterol-rich particles are synthesized in the liver. We found out that PHHC rat fed 1% cholesterol diet accumulates cholesteryl esters (CE) in the liver and also in the VLDL. Acyl-CoA:cholesterol acyltransferase (ACAT) and microsomal triglyceride transfer protein (MTP) may participate in the increased incorporation CE into VLDL. We found out no difference in ACAT and MTP activities in the liver between PHHC rats and control animals. Neither ACAT activity...

Excess production and secretion of very low-density lipoprotein (VLDL) by the liver into the circulatory system is directly related to atherosclerosis, a chronic cardiovascular disease that threatens the lives of many worldwide and continues to be a leading cause of death in the United States. The rate-limiting step in VLDL secretion is its transport from the site of biogenesis, the hepatic endoplasmic reticulum to the cis-Golgi. This step is mediated by a specialized ER- derived vesicle, the VLDL transport vesicle (VTV). Upon exit of the ER the VTV targets, fuses and delivers VLDL into the lumen of the Golgi. The targeting and fusion of the VTV with the Golgi is facilitated by specific set of soluable N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that form a SNARE complex, which is required for the VTV-Golgi fusion and thus delivery to the Golgi. Data from our laboratory indicates that the formation of the SNARE complex requires cytosolic factors. Through the purification of liver cytosol, chromatographic steps, detailed mass spectrometry, immunodepletion and western blotting data it was identified that the protein necessary for SNARE complex formation is Hsc-70. Although Hsc-70's identification is significant, the role it plays in SNARE complex formation for VTV -Golgi fusion is a predicament and yet to be unraveled. In this study we performed a series of co-immunoprecipitation reactions to identify its role in SNARE-complex assembly. Using western blot data we confirmed binding of Hsc-70 with Sec22b, the v-SNARE on the VTV. Moreover, we confirmed the interaction of Hsc-70 with t-SNAREs, (syn5, rBet1 and GOS28) on the Golgi membrane. Removal of Hsc-70 from the liver cytosol resulted in significant reduction of SNARE-complex formation. Ultimately, the identification proteins involved in the process of VLDL delivery to the Golgi would offer therapeutic targets to control VLDL secretion into the blood by the liver.

No description available.