A Targeted Apolipoprotein B-38.9-producing Mutation Causes Fatty Livers in Mice Due to the Reduced Ability of Apolipoprotein B-38.9 to Transport Triglycerides

Abstract

Nonphysiological truncations of apolipoprotein (apo) B-100 cause familial hypobetalipoproteinemia (FHBL) in humans and mice. An elucidation of the mechanisms underlying the FHBL phenotypes may provide valuable information on the metabolism of apo B-containing lipoproteins and the structure-function relationship of apo B. To generate a faithful mouse model of human FHBL, a subtle mutation was introduced into the mouse apo B gene by targeting embryonic stem cells using homologous recombination followed by removal of the selection marker gene by Cre-loxP-mediated site-specific recombination. The engineered mice bear a premature stop codon at residue 1767 and a 42-base pair loxP inserted into intron 24 of the apo B gene, thus closely resembling the apo B-38.9-producing mutation in humans. Apo B-38.9 was the sole apo B protein in homozygote (apob 38.9/38.9 ) plasma. In heterozygotes (apob ؉/38.9 ), apo B-100 and apo B-48 were reduced by 75 and 40%, respectively, and apo B-38.9 represented 20% of total circulating apo B. Hepatic apo B-38.9 mRNA levels were reduced by 40%. In cultured apob ؉/38.9 hepatocytes, apo B-100 was produced in trace quantities, and the synthesis rate of apo B-38.9 relative to apo B-48 was reduced by 40%. However, almost equimolar amounts of apo B-38.9 and apo B-48 were secreted into the media. Pulse-chase studies revealed that apo B-38.9 was secreted at a faster rate and more efficiently than apoB-48. Nevertheless, both apob ؉/38.9 and apob 38.9/38.9 mice had reduced hepatic triglyceride secretion rates and fatty livers. Thus, low mRNA levels or defective secretion of apo B-38.9 may not be responsible for the FHBL phenotypes caused by the apo B-38.9 mutation. Rather, a reduced capacity of apo B-38.9 for triglyceride transport may account for the fatty livers in these mice. Apolipoprotein B (apo B)1 is the major structural protein component of the triglyceride-rich very low density lipoproteins (VLDLs) secreted from the liver and the chylomicrons secreted from the intestine. The full-length apo B (apo B-100) is composed of 4536 amino acid residues (1). Due to a posttranscriptional modification of the apo B gene (apob) mRNA that converts codon 2153, CAA, to a stop codon, UAA, the apo B protein also naturally exists in a truncated form corresponding to the NH 2 -terminal 48% of apo B-100, designated apo B-48 (2, 3). In humans, apo B-48 is produced only by the intestine, whereas both the intestine and the liver in rodents secrete apo B-48 (4, 5). High levels of plasma apo B-containing lipoproteins are a major risk for the development of atherosclerotic diseases. Therefore, mechanisms controlling apo B synthesis and secretion are under active investigation. Nonsense and frameshift mutations in apob that produce nonphysiological COOH-terminal truncations of apo B-100 cause familial hypobetalipoproteinemia (FHBL) in humans, an autosomal codominant disorder characterized by low levels (Ͻ5th percentile) of plasma apo B and low density lipoprotein (LDL) cholesterol (6, 7). Numerous forms of truncated apo...

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

“…The presence of fatty liver was not commented upon in prior reports in which apoB siRNA was delivered in vivo, but a study in which antisense apoB oligonucleotides were used indicated that apoB knockdown did not cause fatty liver (10,21,22,49). However, fatty livers are observed in transgenic mice possessing a mutant apoB gene associated with familial hypobetalipoproteinemia in humans (48). The possibility that apoB knockdown causes fatty liver should be taken into account if apoB is to be considered a therapeutic target in the treatment of hypercholesterolemia in humans.…”

“…Indeed, transgenic mice expressing a truncated form of apoB found in patients with familial hypobetalipoproteinemia, also display a reduced capacity to transport hepatic triglycerides (48). To assess the effects of apoB knockdown on triglyceride transport, we performed oil red staining of liver sections obtained from mice injected with apoB-1 siRNA polyconjugate.…”

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