No abstract

Hypertriglyceridemia is a common pathological condition in humans of mostly unknown etiology. Here we report induction of dyslipidemia characterized by severe hypertriglyceridemia as a result of point mutations in human apolipoprotein A-I (apoA-I). Adenovirus-mediated gene transfer in apoA-I-deficient (apoA-I(-)(/)(-)) mice showed that mice expressing an apoA-I[E110A/E111A] mutant had comparable hepatic mRNA levels with WT controls but greatly increased plasma triglyceride and elevated plasma cholesterol levels. In addition, they had decreased apoE and apoCII levels and increased apoB48 levels in very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL). Fast protein liquid chromatography (FPLC) analysis of plasma showed that most of cholesterol and approximately 15% of the mutant apoA-I were distributed in the VLDL and IDL regions and all the triglycerides in the VLDL region. Hypertriglyceridemia was corrected by coinfection of mice with recombinant adenoviruses expressing the mutant apoA-I and human lipoprotein lipase. Physicochemical studies indicated that the apoA-I mutation decreased the alpha-helical content, the stability, and the unfolding cooperativity of both lipid-free and lipid-bound apoA-I. In vitro functional analyses showed that reconstituted HDL (rHDL) particles containing the mutant apoA-I had 53% of scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux capacity and 37% capacity to activate lecithin:cholesterol acyltransferase (LCAT) as compared to the WT control. The mutant lipid-free apoA-I had normal capacity to promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. The findings indicate that subtle structural alterations in apoA-I may alter the stability and functions of apoA-I and high-density lipoprotein (HDL) and may cause hypertriglyceridemia.

Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for b-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and a-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells.ARTICLE HISTORY

New insights are provided into the role of apoE in cholesterol and triglyceride homeostasis, and of apoA-I in the biogenesis of HDL. Clearance of the lipoprotein remnants and increase in HDL synthesis are obvious targets for therapeutic interventions.

We have investigated the ability of apoE (apolipoprotein E) to participate in the biogenesis of HDL (high-density lipoprotein) particles in vivo using adenovirus-mediated gene transfer in apoA-I-/- (apolipoprotein A-I) or ABCA1-/- (ATP-binding cassette A1) mice. Infection of apoA-I-/- mice with 2x10(9) pfu (plaque-forming units) of an apoE4-expressing adenovirus increased both HDL and the triacylglycerol-rich VLDL (very-low-density lipoprotein)/IDL (intermediate-density lipoprotein)/LDL (low-density lipoprotein) fraction and generated discoidal HDL particles. ABCA1-/- mice treated similarly failed to form HDL particles, suggesting that ABCA1 is essential for the generation of apoE-containing HDL. Combined infection of apoA-I-/- mice with a mixture of adenoviruses expressing both apoE4 (2x10(9) pfu) and human LCAT (lecithin:cholesterol acyltransferase) (5x10(8) pfu) cleared the triacylglycerol-rich lipoproteins, increased HDL and converted the discoidal HDL into spherical HDL. Similarly, co-infection of apoE-/- mice with apoE4 and human LCAT corrected the hypercholesterolaemia and generated spherical particles, suggesting that LCAT is essential for the maturation of apoE-containing HDL. Overall, the findings indicate that apoE has a dual functionality. In addition to its documented functions in the clearance of triacylglycerol-rich lipoproteins, it participates in the biogenesis of HDL-sized apoE-containing particles. HDL particles generated by this pathway may account at least for some of the atheroprotective functions of apoE.

Preadipocyte differentiation capacity declines between middle and old age. Expression of the adipogenic transcription factors, CCAAT/enhancer-binding protein (C/EBP) ␣ and peroxisome proliferator-activated receptor ␥ (PPAR␥), is lower in differentiating preadipocytes from old than young animals, although no age-related changes occur in C/EBP␤ mRNA, which is upstream of C/EBP␣ and PPAR␥. C/EBP␤-liver-enriched inhibitory protein (C/EBP␤-LIP), a truncated C/EBP␤ isoform that is a dominant inhibitor of differentiation, increases with aging in rat fat tissue and preadipocytes. CUG triplet repeat-binding protein-1 (CUGBP1) binds to C/EBP␤ mRNA, increasing C/EBP␤-LIP translation. Abundance and nucleotide binding activity of CUGBP1 increased with aging in preadipocytes. CUGBP1 overexpression in preadipocytes from young animals increased C/EBP␤-LIP and impaired adipogenesis. Decreasing CUGBP1 in preadipocytes from old rats by RNA interference reduced C/EBP␤-LIP abundance and promoted adipogenesis. Tumor necrosis factor-␣, levels of which are elevated in fat tissue with aging, increased CUGBP1 protein, CUGBP1 binding activity, and C/EBP␤-LIP in preadipocytes from young rats. Thus, CUGBP1 contributes to regulation of adipogenesis in primary preadipocytes and is responsive to tumor necrosis factor-␣. With aging, preadipocyte CUGBP1 abundance and activity increases, resulting in enhanced translation of the C/EBP␤-LIP isoform, thereby blocking effects of adipogenic transcription factors, predisposing preadipocytes from old animals to resist adipogenesis. Altered translational processing, possibly related to changes in cytokine milieu and activation of stress responses, may contribute to changes in progenitor differentiation and tissue function with aging.

Obesity and its related pathologies constitute a major cause of death, with rates increasing at an alarming pace [1]. By the beginning of the millennium, overweight adults accounted for over 15% of the world's population (body mass index > 30, World Health Organization) [2], with this number increasing to 50% within the USA and Europe [3]. Obesity develops as a result of disruption of the homeostasis between food intake and energy expenditure, and therefore factors affecting these processes are the focus of extensive Obesity is a central feature of the metabolic syndrome and is associated with increased risk for insulin resistance and type II diabetes. Here, we investigated the contribution of human apoliprotein E3 and mouse apoliprotein E to the development of diet-induced obesity in response to western-type diet. Our data show that apolipoprotein E contributes to the development of obesity and other related metabolic disorders, and that human apolipoprotein E3 is more potent than mouse apolipoprotein E in promoting obesity in response to western-type diet. Specifically, we found that apolipoprotein E3 knock-in mice fed western-type diet for 24 weeks became obese and developed hyperglycemia, hyperinsulinemia, hyperleptinemia, glucose intolerance and insulin resistance that were more severe than in C57BL/6 mice. In contrast, apolipoprotein E-deficient mice fed westerntype diet for the same period were resistant to diet-induced obesity, had normal plasma glucose, leptin and insulin levels, and exhibited normal responses to glucose tolerance and insulin resistance tests. Furthermore, low-density lipoprotein receptor-deficient mice were more sensitive to the development of diet-induced obesity and insulin resistance than apolipoprotein E-deficient mice, but were still more resistant than C57BL/6 mice, raising the possibility that low-density lipoprotein receptor mediates, at least in part, the effects of apolipoprotein E on obesity. Taken together, our findings suggest that, in addition to other previously identified mechanisms of obesity, apolipoprotein E and possibly the chylomicron pathway are also important contributors to the development of obesity and related metabolic dysfunctions in mice. , LDLrdeficient; LpL, lipoprotein lipase; VLDL, very low-density lipoprotein.

We have studied the contribution of the carboxy terminal domains of lipid-free apoE isolated from apoE-expressing cell cultures in binding to phospholipids and have determined the affinities of reconstituted POPC-apoE particles for the apoER2. It was found that the initial rate of association of apoE2, apoE3, apoE4, and a mutant form apoE4R158M to multilamellar DMPC vesicles was similar and was reduced and eventually diminished by gradual deletion of the carboxy terminal segments. The truncated apoE forms retained their ability to associate with plasma lipoproteins. Receptor binding studies were performed using the ldlA-7 cells expressing apoER2 and transiently transfected COS-M6 and the appropriate control untransfected cells. Specific binding to apoER2 was obtained by subtracting from the total binding to the receptor-expressing cells the nonspecific binding values of the untransfected cells. POPC-apoE particles generated using apoE3, apoE4, the truncated apoE4-259, apoE4-229, apoE4-202, and apoE-165, and the mutant apoE4R158M all bound tightly to the apoER2 (K(d) range of 12 +/- 3 to 19 +/- 4 microg/mL). POPC-apoE2 bound with reduced affinity (K(d) = 31 +/- 5.3 microg/mL). The findings establish that the apoER2 binding domain of apoE is in the 1-165 amino terminal region, whereas the carboxy terminal 230-299 region of apoE is required for efficient initial association with phospholipids.