The peroxisome proliferator-activated receptor (PPAR) ␣ agonist WY 14,643 increased the secretion of apolipoprotein (apo) B-100, but not that of apoB-48, and decreased triglyceride biosynthesis and secretion from primary rat hepatocytes. These effects resulted in decreased secretion of apoB-100-very low density lipoprotein (VLDL) and an increased secretion of apoB-100 on low density lipoproteins/intermediate density lipoproteins. ApoB-48-VLDL was also replaced by more dense particles. The proteasomal inhibitor lactacystin did not influence the recovery of apoB-100 or apoB-48 in primary rat hepatocytes, indicating that co-translational (proteasomal) degradation is of less importance in these cells. Treatment with WY 14,643 made the recovery of apoB-100 sensitive to lactacystin, most likely reflecting the decreased biosynthesis of triglycerides. The PPAR␣ agonist induced a significant increase in the accumulation of pulse-labeled apoB-100 even after a short pulse (2-5 min). There was also an increase in apoB-100 nascent polypeptides, indicating that the co-translational degradation of apoB-100 was inhibited. However, a minor influence on an early posttranslation degradation cannot be excluded. This decreased co-translational degradation of apoB-100 explained the increased secretion of the protein. The levels of apoB-48 remained unchanged during these pulse-chase experiments, and albumin production was not affected, indicating a specific effect of PPAR␣ agonists on the co-translational degradation of apoB-100. These findings explain the difference in the rate of secretion of the two apoB proteins seen after PPAR␣ activation. PPAR␣ agonists increased the expression and biosynthesis of liver fatty acidbinding protein (LFABP). Increased expression of LFABP by transfection of McA-RH7777 cells increased the secretion of apoB-100, decreased triglyceride biosynthesis and secretion, and increased PPAR␣ mRNA levels. These findings suggest that PPAR␣ and LFABP could interact to amplify the effect of endogenous PPAR␣ agonists on the assembly of VLDL.The peroxisome proliferator-activated receptor (PPAR) 1 ␣ has a central role in the regulation of lipid metabolism, and unsaturated long-chain fatty acids (LCFAs) are among the natural ligands for this receptor (1). PPAR␣ is also activated by so-called peroxisome proliferators, a group of compounds that includes the lipid-lowering fibrates (1, 2). The activation of PPAR␣ results in important and diverse effects on lipid metabolism, such as an increase in the transcription of genes involved in mitochondrial and peroxisomal ␤-oxidation, and the regulation of the expression of apolipoprotein (apo) A-I, apoC-III (2), and PPAR␣ itself (3). Moreover, PPAR␣ regulates the transcription of LFABP, and the LFABP gene promotor has a PPAR-responsive element (2, 4). It has been shown that both fibrates and unsaturated LCFAs increase the gene expression and production of LFABP (5-8).LFABP is an abundant cytosolic protein present in the liver and intestine. The protein binds LCFAs and their CoA-e...

thesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 cells. In this system, 10 -15% of the apoB-100 pool was converted to VLDL in a time-and temperature-dependent way. The assembly process was highly dependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL. Brefeldin A inhibited VLDL assembly in this system, as did a synthetic peptide (corresponding to Nterminal amino acids 2-17 of ARF-1) that displaces ARF-1 from the membrane. Thus, active ARF-1 is also needed for cell-free assembly of VLDL. Guanosine 5-3-O-(thio)triphosphate also inhibited VLDL assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.

The liver plays a primary role in lipid metabolism. Important functions include the synthesis and incorporation of hydrophobic lipids, triacylglycerols and cholesteryl esters into the core of water-miscible particles called lipoproteins and the secretion of these particles into the circulation for transport to distant tissues. In this article, we present a brief overview of one aspect of the assembly process of very low density lipoproteins, namely, possible mechanisms for combining core lipids with apolipoprotein B. This is a complex process in which apolipoprotein B interacts with core lipids to form very low density lipoproteins by a two-step process that can be dissociated biochemically.

We have investigated the influence of structures in the tRNA anticodon loop and stem on the ability of the anticodon to discriminate among codons. We had previously shown that anticodon UCC, when placed in the structural context of tRNAG?y from Escherichia coli, discriminated efficiently between the glycine codons, as required by the wobble rules. Thus, this anticodon read GGA and GGG but did not read GGU and GGC, whereas in mycoplasma tRNAGIY, the same anticodon did not discriminate among the glycine codons. We have now determined the reading properties of three constructions based on tRNAlGly containing the anticodon UCC in different structural contexts. In one of these constructs, tRNAlIY-ASL, the anticodon loop and stem are the same as in mycoplasma tRNAGIY. The second construct, tRNAG,'-AS, has an anticodon stem identical with the mycoplasma tRNAGIy, whereas in the last construct, tRNAGly-C32 the only difference from tRNA?GY(UCC) is that the uridine in position 32 of the anticodon loop has been replaced by cytidine. These constructs were tested for ability to read glycine codons in an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each codon. We found that the anticodon UCC, when present in tRNAG'Y-AS, discriminated among the glycine codons, whereas in the constructs tRNAGI1-ASL and tRNA ¶'Y-C32, the same anticodon had lost its ability to discriminate-i.e., it behaved as in mycoplasma tRNAGIY. These results strongly suggest that nt 32 of the anticodon loop of tRNAO'Y(UCC) decisively influences the reading properties of the anticodon UCC.Analysis of tRNA genes and their gene products in mitochondria (1-3) has revealed that in these organelles each family box (group of four codons that have the same nucleotides in the first two positions and that all denote the same amino acid) is read by only one tRNA, which cannot discriminate among the nucleotides in the third-codon position, as required by the wobble rules (4). A similar situation exists in chloroplasts (5, 6), although in this case only about half of the codon families are read without discrimination. Our own analysis of Mycoplasma mycoides indicated that in this microorganism the family boxes, except for the threonine box, are read by only one tRNA each (7-9). The same results have been obtained in Mycoplasma capricolum by Andachi et al. (10). tRNAs that read without discrimination among nucleotides in the third-codon position, in most cases, have an unsubstituted uridine as their wobble nucleotide.These results are reminiscent of a phenomenon we had previously observed in a protein-synthesizing in vitro system programmed with the phage messenger MS2 RNA. In this system we found that family boxes could be read by a single tRNA with an undiscriminating method we referred to as "reading by two-out-of-three" (11). The capacity for this unconventional type of reading varied among different isoaccepting tRNAs. The tRNAGly(UCC) from M. mycoides (12) was almost as efficient in the unconventional reading of cod...

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