Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory elementbinding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4 -5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulinmediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 M T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia.Autosomal dominant hypercholesterolemia is associated with mutations in genes involved in the regulation of LDL 2 homeostasis. The most common and severe form of monogenic hypercholesterolemia is familial hypercholesterolemia, caused by mutations in the LDL receptor (LDLr) (1). Familial hypercholesterolemia is characterized by elevated plasma LDL-cholesterol levels and premature cardiovascular disease.Another form of this disease, familial defective apolipoprotein (apo)B100, is caused by mutations in the LDLr binding domain of ApoB100 (1). ApoB100 is synthesized by the liver and is the major protein component of very low density lipoprotein and LDL. Recently, proprotein convertase subtilisin kexin 9 (PCSK9) has been identified as the third gene involved in autosomal dominant hypercholesterolemia (5). Proprotein convertases are proteolytic enzymes that cleave their substrate, producing a biologically active molecule (2). We showed that patients mutated in PCSK9 prodomain (mutation S127R) have decreased LDL catabolism and increased very low density lipoprotein, intermediary density lipoprotein, and LDL production (3). Studies on knock-out mice and plasma lipid profiles of patients with nonsense mutations, showed that PCSK9 deficiency results in low circulating LDL-cholesterol concentrations (4 -6). Althou...

Vitamin E is a lipophilic anti-oxidant that can prevent the oxidative damage of atherogenic lipoproteins. However, human trials with vitamin E have been disappointing, perhaps related to ineffective levels of vitamin E in atherogenic apoB-containing lipoproteins. Phospholipid transfer protein (PLTP) promotes vitamin E removal from atherogenic lipoproteins in vitro, and PLTP deficiency has recently been recognized as an antiatherogenic state. To determine whether PLTP regulates lipoprotein vitamin E content in vivo, we measured ␣-tocopherol content and oxidation parameters of lipoproteins from PLTP-deficient mice in wild type, apoEdeficient, low density lipoprotein (LDL) receptor-deficient, or apoB/cholesteryl ester transfer protein transgenic backgrounds. In all four backgrounds, the vitamin E content of very low density lipoprotein (VLDL) and/or LDL was significantly increased in PLTP-deficient mice, compared with controls with normal plasma PLTP activity. Moreover, PLTP deficiency produced a dramatic delay in generation of conjugated dienes in oxidized apoB-containing lipoproteins as well as markedly lower titers of plasma IgG autoantibodies to oxidized LDL. The addition of purified PLTP to deficient plasma lowered the vitamin E content of VLDL plus LDL and normalized the generation of conjugated dienes. The data show that PLTP regulates the bioavailability of vitamin E in atherogenic lipoproteins and suggest a novel strategy for achieving more effective concentrations of antioxidants in lipoproteins, independent of dietary supplementation.The oxidation theory of atherogenesis has received wide support from a number of different lines of evidence (1, 2). In particular, treatment of hypercholesterolemic animals with a variety of potent synthetic anti-oxidants has resulted in inhibition of the progression of atherosclerosis (3). However, a direct relationship between the susceptibility of LDL 1 to oxidation and the extent of atherosclerosis has not been found in all studies, and attempts to prevent atherogenesis by feeding diets enriched in "natural" anti-oxidants have provided mixed and sometimes disappointing results (2, 3). Recently, it was shown that feeding large doses of vitamin E to apoE-deficient mice decreased the progression of atherosclerosis (4, 5). However, with a few exceptions (6, 7), the administration of vitamin E in human trials has been negative (8 -12). An important issue that has not been addressed in such studies is the actual concentrations of vitamin E in atherogenic lipoproteins. Recently, mice with ␣-tocopherol transfer protein deficiency were shown to have reduced vitamin E content in lipoproteins, and moderately increased susceptibility to atherosclerosis (13). However, little is known of the physiological mechanism regulating the turnover and levels of vitamin E in the plasma lipoproteins.The plasma phospholipid transfer protein (PLTP) mediates both net transfer and exchange of phospholipids between lipoproteins (14). PLTP can also bind and transfer several other amphipathic lipids, ...

ABCA1, the mutant molecule in Tangier

Mutations in Proprotein Convertase SubtilisinKexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.