Background-LDL receptor-deficient "apolipoprotein (apo)-B100 -only" mice (Ldlr Ϫ/Ϫ Apob 100/100 have elevated LDL cholesterol levels on a chow diet and develop severe aortic atherosclerosis. We hypothesized that both the hypercholesterolemia and the susceptibility to atherosclerosis could be eliminated by switching off hepatic lipoprotein production. Methods and Results-We bred LdlrϪ/Ϫ Apob 100/100 mice that were homozygous for a conditional allele for Mttp (the gene for microsomal triglyceride transfer protein) and the inducible Mx1-Cre transgene. In these animals, which we called "Reversa mice," the hypercholesterolemia could be reversed, without modifying the diet or initiating a hypolipidemic drug, by the transient induction of Cre expression in the liver. After Cre induction, hepatic Mttp expression was virtually eliminated (as judged by quantitative real-time PCR), hepatic lipoprotein secretion was abolished (as judged by electron microscopy), and LDLs were virtually eliminated from the plasma. Intestinal lipoprotein production was unaffected. In mice fed a chow diet, Cre induction reduced plasma cholesterol levels from 233.9Ϯ46.0 to 37.2Ϯ6.5 mg/dL. In mice fed a high-fat diet, cholesterol levels fell from 525.7Ϯ32.2 to 100.6Ϯ14.3 mg/dL. The elimination of hepatic lipoprotein production completely prevented both the development of atherosclerosis and the changes in gene expression that accompany atherogenesis. Conclusions-We developed mice in which hypercholesterolemia can be reversed with a genetic switch. These mice will be useful for understanding gene-expression changes that accompany the reversal of hypercholesterolemia and atherosclerosis.

Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo posttranslational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by Rce1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking Rce1 in the heart, an organ where Rce1 is expressed at particularly high levels. The hearts from heart-specific Rce1 knockout mice manifested reduced levels of both the Rce1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific Rce1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, ϳ70% of the mice had died. Pathological studies revealed that the heartspecific Rce1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific Rce1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.Proteins terminating with a CAAX 1 motif, such as the nuclear lamins and the Ras and Rho proteins, undergo three sequential post-translational processing steps (1-4). First, the C-terminal cysteine (i.e. the C of the CAAX motif) is "lipidated" with a 15-carbon farnesyl or a 20-carbon geranylgeranyl lipid. This processing step is carried out by cytosolic prenyltransferases (protein farnesyltransferase and protein geranylgeranyltransferase type I). Second, a prenylprotein-specific endoprotease of the endoplasmic reticulum, Ras-converting enzyme 1 (Rce1), clips off the last three amino acids from the protein (i.e. the -AAX) (5-7). Third, the newly exposed C-terminal isoprenylcysteine is methylesterified by isoprenylcysteine carboxyl methyltransferase (Icmt), a prenylprotein-specific, S-adenosylmethionine-dependent methyltransferase of the endoplasmic reticulum (8 -11).Prenylation of CAAX proteins is vitally important to eukaryotic cells (1-4). Yeast lacking the shared ␣-chain of farnesyltransferase and geranylgeranyltransferase type I are not viable (12). In mammalian cells, prenylation of CAAX proteins is absolutely required for their proper targeting to membrane surfaces and for proper protein function (1-4). The importance of protein farnesylation is clearly indicated by the fact that mice lacking farnesyltransferase die early during embryonic development (before embryonic day 6.5). 2 The geranylgeranylation of CAAX proteins is also critical for normal cellular function. Drugs that inhibit geranylgeranylation of CAAX proteins have pronounced effects on cell growth and can trigger apoptosis (13-15). Further underscoring the im...

a-Tocopherol is a lipid-soluble antioxidant that helps to prevent oxidative damage to cellular lipids. aTocopherol is absorbed by the intestine and is taken up and retained by the liver; it is widely presumed that a-tocopherol is then delivered to peripheral tissues by the secretion of VLDL. To determine whether VLDL secretion is truly important for the delivery of a-tocopherol to peripheral tissues, we examined a-tocopherol metabolism in mice that lack microsomal triglyceride transfer protein (Mttp) expression in the liver and therefore cannot secrete VLDL (Mttp D/D mice). Mttp D/D mice have low plasma lipid levels and increased stores of lipids in the liver. Similarly, a-tocopherol levels in the plasma were lower in Mttp D/D mice than in controls, whereas hepatic a-tocopherol stores were higher. However, a-tocopherol levels in the peripheral tissues of Mttp D/D mice were nearly identical to those of control mice, suggesting that VLDL secretion is not critical for the delivery of atocopherol to peripheral tissues. When fed a diet containing deuterated a-tocopherol, Mttp D/D and control mice had similar incorporation of deuterated a-tocopherol into plasma and various peripheral tissues. We conclude that the absence of VLDL secretion has little effect on the stores of a-tocopherol in peripheral tissues, at least in the mouse. Vitamin E (tocopherols and tocotrienols) is a lipidsoluble antioxidant that helps to prevent oxidative damage to polyunsaturated fatty acids (1, 2). In the setting of vitamin E deficiency, neurodegeneration occurs and fertility is impaired (2, 3). Among the four main tocopherol isoforms (a, g, d, b), a-tocopherol is present in the highest concentrations, both in the plasma and in peripheral tissues (4-6). The a-tocopherol in the plasma is carried exclusively within the plasma lipoproteins (7, 8). Because vitamin E is an antioxidant and the oxidation of lipoproteins has been linked to atherogenesis, vitamin E has attracted interest as a potential therapy to prevent atherosclerosis (9, 10).Dietary vitamin E is absorbed in the intestine and transported into the circulation by the intestinal triglyceride-rich lipoproteins (chylomicrons) (11,12). During the hydrolysis of triglycerides by lipoprotein lipase, some tocopherols are probably transferred to other lipoproteins and/or taken up by peripheral tissues. However, most of the tocopherols are thought to be delivered to the liver by chylomicron remnants (11). Within the liver, a cytosolic protein, a-tocopherol transfer protein (a-TTP), selectively binds and retains a-tocopherol, whereas the other tocopherol isoforms are metabolized and/or secreted into the bile. a-TTP binding of a-tocopherol in the liver is physiologically important, as a genetic defect in a-TTP causes vitamin E deficiency in humans. Importantly, a-TTP has been thought to mediate the transfer of a-tocopherol for packaging into VLDL for delivery to peripheral tissues (13). The transport of tocopherol in rodents and humans is likely quite similar, even though rodents are ...