Specific sequences in the N and C termini of apolipoprotein A-IV modulate its conformation and lipid association

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.     

Structural properties and lipid binding of human apolipoprotein A-IV

The in vivo affinity of human apolipoprotein A-IV (apo-A-IV) for plasma lipoproteins is considerably less than that of other apolipoproteins. We have therefore studied its spectroscopic properties and its association with model chylomicrons to investigate its structural characteristics and to define their influence upon its affinity for lipids. Fluorescence emission spectra of apo-A-IV in dilute aqueous solution revealed that its single tryptophan residue resides in a pH-sensitive hydrophobic domain, which is maximally protected from iodide quenching at pH 7.5. Denaturation of apo-A-IV by guanidine hydrochloride caused a multiphasic fluorescence emission red shift, with an unusual enhancement of quantum yield. Circular dichroism spectroscopy of apo-A-IV demonstrated negative ellipticity maxima at 210 and 222 nm, consistent with 54% alpha-helical structure. The alpha-helicity of apo-A-IV as measured by [theta]222 was also pH-sensitive and displayed a distinctive decrease between pH 7.0 and 8.0. Apo-A-IV was exquisitely sensitive to denaturation by guanidine hydrochloride, and its estimated free energy of stabilization in aqueous solution was near zero. Apo-A-IV bound to the surface of Sf greater than 400 particles of a phospholipid-triglyceride emulsion in a noncooperative, concentration-dependent manner. The affinity of apo-A-IV for these model chylomicrons was influenced by changes in pH or addition of guanidine hydrochloride in a manner which correlated well with the structural changes observed under similar conditions. We conclude that human apolipoprotein A-IV possesses several biophysical properties characteristic of the better studied plasma apolipoproteins, yet, apo-A-IV appears to be marginally stable in aqueous solution and its structural characteristics and lipid binding properties are particularly sensitive to environment.     

Lipoprotein ApoC-II activation of lipoprotein lipase. Modulation by apolipoprotein A-IV

Lipoprotein lipase (LPL)-mediated hydrolysis of triglycerides (TG) contained in chylomicrons requires the presence of a cofactor, apolipoprotein (apo) C-II. The physiological mechanism by which chylomicrons gain apoC-II necessary for LPL activation in whole plasma is not known. Using a gum arabic stabilized TG emulsion, activation of LPL by lipoprotein apoC-II was studied. Hydrolysis of TG by LPL was greater in the presence of serum than with addition of either high density lipoproteins (HDL) or very low density lipoproteins (VLDL). LPL activation by either VLDL or HDL increased with addition of the lipoprotein-free fraction of plasma. A similar increase in LPL activity by addition of the lipoprotein-free fraction together with HDL or VLDL was observed when another TG emulsion (Intralipid) or TG-rich lipoproteins from an apoC-II deficient subject were used as a substrate. Human apoA-IV, apoA-I, apoE, and cholesteryl ester transfer protein were assessed for their ability to increase LPL activity in the presence of VLDL. At and below physiological concentrations, only apoA-IV increased LPL activity. One hundred percent of LPL activity measured in the presence of serum was achieved using VLDL plus apoA-IV. In the absence of an apoC-II source, apoA-IV had no effect on LPL activity. Removal of greater than 80% of the apoA-IV from the nonlipoprotein-containing fraction of plasma by incubation with Intralipid markedly reduced its ability to activate LPL in the presence of VLDL or HDL. Gel filtration chromatography demonstrated that incubation of the nonlipoprotein-containing fraction of plasma with HDL and the TG emulsion caused increased transfer of apoC-II to the emulsion and association of apoA-IV with HDL. Our studies demonstrate that apoA-IV increases LPL activation in the presence of lipoproteins. We hypothesize that apoA-IV is required for efficient release of apoC-II from either HDL or VLDL, which then allows for LPL-mediated hydrolysis of TG in nascent chylomicrons.     

Enthalpy-driven apolipoprotein A-I and lipid bilayer interaction indicating protein penetration upon lipid binding

The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K(a)), the number of protein molecules bound per vesicle (n), enthalpy change (DeltaH degrees), entropy change (DeltaS degrees ), and the heat capacity change (DeltaC(p) degrees). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 degrees C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the alpha-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of POPC/FC at a molar ratio of 6/1 at (2.8-4) x 10(6) M(-1) and 2.4 apoA-I molecules bound per vesicle or 1.4 x 10(2) phospholipids per bound apoA-I. A plot of DeltaH degrees against temperature displayed a linear behavior, from which the DeltaC(p) degrees per mole of bound apoA-I was calculated to be -2.73 kcal/(mol x K). These results suggested that binding of apoA-I to POPC vesicles is characterized by nonclassical hydrophobic interactions, with alpha-helix formation as the main driving force for the binding to cholesterol-containing vesicles. In addition, comparison to literature data on peptides suggested a cooperativity of the helices in apoA-I in lipid interaction.     

Sequence similarity between protein B and human apolipoprotein A-IV

Sequence comparison of protein B (CAMP-factor) with human apolipoprotein A-IV (apo A-IV) revealed 32% similarity between the N-terminal part of protein B and a part of the putative lipid-binding domain of apo A-IV. The significance of this similarity is discussed with respect to the structure/function relationship of protein B.     

Apolipoprotein A-IV polymorphism and its effect on plasma lipid and apolipoprotein concentrations

Recently, we determined the apolipoprotein E (apoE) phenotype distribution in 2,000 randomly selected 35-year-old male individuals by slab gel isoelectric focusing of delipidated plasma samples, followed by immunoblotting using anti-apoE antiserum. These blots have been successfully re-used for immunovisualization of apoA-IV isoelectric focusing patterns. In a population sample of 1,393 individuals, four distinct apoA-IV isoforms were detected, encoded by the alleles A-IV*0, A-IV*1, A-IV*2, and A-IV*3 with gene frequencies of 0.002, 0.901, 0.079, and 0.018, respectively. The mean of plasma cholesterol, triglyceride, apoB and E levels did not differ significantly among the different apoA-IV phenotype groups. For these lipoprotein parameters, less than 0.1% of the total phenotypic variance could be accounted for by the APOA-IV gene locus. Our results did not show any effect of apoA-IV polymorphism on plasma apoA-I levels nor could we find any correlation between plasma levels of apoA-I and apoA-IV within the different apoA-IV phenotype groups. The plasma level of apoA-IV in subjects bearing the A-IV*3 allele is significantly lower than in subjects without the A-IV*3 allele (5 mg/dl versus 14 mg/dl). We therefore conclude that, in contrast to the apoE polymorphism, the polymorphism at the APOA-IV locus does not influence any of the levels of the lipoprotein parameters considered except apoA-IV.     

Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid

Membrane lipids have been implicated to influence the activity of G-protein-coupled receptors (GPCRs). Almost all of our knowledge on the role of lipids on GPCR and G protein function comes from work on the visual pigment rhodopsin and its G protein transducin, which reside in a highly specialized membrane environment. Thus, insight gained from rhodopsin signaling may not be simply translated to other nonvisual GPCRs. Here, we investigated the effect of lipid head group charges on the signal transduction properties of the class A GPCR neurotensin (NT) receptor 1 (NTS1) under defined experimental conditions, using self-assembled phospholipid nanodiscs prepared with the zwitter-ionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), or a POPC/POPG mixture. A combination of dynamic light scattering and sedimentation velocity showed that NTS1 was monomeric in POPC-, POPC/POPG-, and POPG-nanodiscs. Binding of the agonist NT to NTS1 occurred with similar affinities and was essentially unaffected by the phospholipid composition. In contrast, Gq protein coupling to NTS1 in various lipid nanodiscs was significantly different, and the apparent affinity of Gαq and Gβ(1)γ(1) to activated NTS1 increased with increasing POPG content. NTS1-catalyzed GDP/GTPγS nucleotide exchange at Gαq in the presence of Gβ(1)γ(1) and NT was crucially affected by the lipid type, with exchange rates higher by 1 or 2 orders of magnitude in POPC/POPG- and POPG-nanodiscs, respectively, compared to POPC-nanodiscs. Our data demonstrate that negatively charged lipids in the immediate vicinity of a nonvisual GPCR modulate the G-protein-coupling step.     

Frequency and effect of human apolipoprotein A-IV polymorphism on lipid and lipoprotein levels in an Icelandic population

Summary Human apolipoprotein A-IV (apo A-IV) exhibits a genetic polymorphism with two common alleles, A-IV¹ and A-IV², in Caucasian populations. We have investigated this polymorphism in the Icelandic population. The frequencies of the two alleles are significantly different from middel European populations with a higher frequency of the A-IV² allele (0.117 versus 0.077) occurring in Iceland. The alleles at the apo A-IV locus have significant effects on plasma high density lipoprotein cholesterol (HDL-C) and triglyceride levels. The average effect of the A-IV² allele is to raise HDL-C by 4.9 mg/dl and to lower triglyceride levels by 19.4mg/dl. We estimate that the genetic variability at the apo A-IV gene locus accounts for 3.1% of the total variability of HDL-C and for 2.8% of the total variability of triglycerides in the population from Iceland. This confirms and extends our previous observations on apo A-IV allele effects in Tyroleans in an independent population.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

Adaptation of intestinal production of apolipoprotein A-IV during chronic feeding of lipid

We examined the effect of daily fat supplementation on intestinal gene expression and protein synthesis and plasma levels of apolipoprotein A-IV (apo A-IV). Rats were fasted overnight and then given intragastric bolus infusion of either saline or fat emulsion after 0, 1, 2, 4, 8, or 16 days of similar daily feedings. Four hours after the final saline or fat infusion, plasma and jejunal mucosa were harvested; plasma levels of apo A-IV, triglycerides, and leptin were measured, as well as mucosal apo A-IV mRNA levels and biosynthesis of apo A-IV protein. In response to fat, plasma apo A-IV showed an initial 40% increase compared with saline-injected control rats; with continued daily fat feeding, the plasma A-IV response showed rapid and progressive diminution such that by 4 days, plasma A-IV was not different between fat- and saline-fed groups. Jejunal mucosal apo A-IV synthesis and mRNA levels also showed time-dependent refractoriness to fat feeding. However, the kinetics of this effect were considerably slower than in the case of plasma, requiring 16 days for completion. There was no correlation between plasma leptin or triglyceride levels and intestinal apo A-IV synthesis or plasma apo A-IV. These results indicate rapid, fat-induced, posttranslational adapation of plasma apo A-IV levels and a slower, but similarly complete pretranslational adaptation of intestinal apo A-IV production, which are independent of plasma levels of leptin.     

Human apolipoprotein A-IV polymorphism: frequency and effect on lipid and lipoprotein levels

Summary Human apolipoprotein (apo) A-IV is genetically polymorphic, the apo A-IV polymorphism being controlled by two common alleles, A-IV¹ and A-IV². We have developed a method for typing the apo A-IV polymorphism by Western blotting using polyclonal rabbit antiapo A-IV as the first and gold-labeled antirabbit IgG as the second antibody. Apolipoprotein phenotypes were determined in plasma samples from 473 tiroleans. The frequencies of the apo A-IV alleles in this sample were f(A-IV¹)=0.919, f(A-IV²)=0.077, and f(A-IV³)=0.004. Although average triglyceride levels were lower in apo A-IV 2-1 heterozygotes, average total serum cholesterol and triglyceride levels were not significantly different among apo A-IV types. High density lipoprotein (HDL) cholesterol was significantly increased in individuals with the A-IV 2-1 phenotype. We estimate that genetic variation at the apo A-IV gene locus accounts for 11% of the total variability in HDL-cholesterol levels in Tiroleans. The effects of the apo A-IV polymorphism described here are consistant with, and may serve to enrich, our limited knowledge of the role of apo A-IV in lipid metabolism.     

Modulation of the lipid binding properties of the N-terminal domain of human apolipoprotein E3.

Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low-density lipoprotein (LDL) receptor family. Receptor recognition is mediated by residues 130-150 in the independently folded, 22-kDa N-terminal (NT) domain. This elongated globular four-helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface. Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C. Likewise, in dimyristoylglycerophosphocholine (Myr2Gro-PCho) vesicle transformation assays, 100 microg apoE3 NT induced only 15% reduction in vesicle (250 microg) light scattering intensity after 30 min. ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro-PCho vesicles was concentration-dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength. In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT-mediated vesicle transformation rates were enhanced > 10-fold compared with Myr2Gro-PCho and activity decreased with increasing buffer ionic strength. Solution pH had a dramatic effect on the kinetics of apoE3 NT-mediated Myr2Gro-PCho vesicle transformation with increased rates observed as a function of decreasing pH. Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0. Similarly, fluorescent dye binding experiments with 8-anilino-1-naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH. These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure.     

Modulation of L-type Ca2+ channels by gbeta gamma and calmodulin via interactions with N and C termini of alpha 1C

Neuronal voltage-dependent Ca(2+) channels of the N (alpha(1B)) and P/Q (alpha(1A)) type are inhibited by neurotransmitters that activate G(i/o) G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gbetagamma subunit of G proteins to the channel. Since cardiac and neuronal L-type (alpha(1C)) voltage-dependent Ca(2+) channels are not modulated in this way, they are presumed to lack interaction with Gbetagamma. However, here we demonstrate that both Gbetagamma and calmodulin directly bind to cytosolic N and C termini of the alpha(1C) subunit. Coexpression of Gbetagamma reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca(2+), and is eliminated by EGTA. The N and C termini of alpha(1C) appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gbetagamma. Deletion of the N terminus profoundly impairs the Ca(2+)/calmodulin-dependent inactivation. We propose that Gbetagamma and calmodulin regulate the L-type Ca(2+) channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of alpha(1C).     

Regulation of intestinal and hypothalamic apolipoprotein A-IV

This review discusses the regulation of the intestinal and hypothalamic apolipoprotein A-IV (apo A-IV) gene and protein expression. Apo A-IV is a glycoprotein secreted together with triglyceride-rich lipoproteins by the small intestine. Intestinal apo A-IV synthesis is stimulated by fat absorption, probably mediated by chylomicron formation. This stimulation of intestinal apo A-IV synthesis is attenuated by intravenous leptin infusion. Chronic ingestion of a high-fat diet blunts the intestinal apo A-IV in response to dietary lipid. Intestinal apo A-IV synthesis is also stimulated by members of the pancreatic polypeptide family, including peptide YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP). Recently, apo A-IV was demonstrated to be present in the hypothalamus as well. Hypothalamic apo A-IV level was reduced by food deprivation and restored by lipid feeding. Intracerebroventricular administration of apo A-IV antiserum stimulated feeding and decreased the hypothalamic apo A-IV mRNA level, implying that feeding is intimately regulated by endogenous hypothalamic apo A-IV. Central administration of NPY significantly increased hypothalamic apo A-IV mRNA levels in a dose-dependent manner.     

Overexpression of apolipoprotein A-IV enhances lipid transport in newborn swine intestinal epithelial cells

Apolipoprotein A-IV (apoA-IV) has myriad functions, including roles as a post-prandial satiety factor and lipid antioxidant. ApoA-IV is expressed in mammalian small intestine and is up-regulated in response to lipid absorption. In newborn swine jejunum, a high fat diet acutely induces a 7-fold increase in apoA-IV expression. To determine whether apoA-IV plays a role in the transport of absorbed lipid, swine apoA-IV was overexpressed in a newborn swine enterocyte cell line, IPEC-1, followed by analysis of the expression of genes related to lipoprotein assembly and lipid transport, as well as quantitation of lipid synthesis and secretion. A full-length swine apoA-IV cDNA was cloned, sequenced, and inserted into a Vp and Rep gene-deficient adeno-associated viral vector, containing the cytomegalovirus immediate early promoter/enhancer and neomycin resistance gene, and was used to transfect IPEC-1 cells. Control cells were transfected with the same vector minus the apoA-IV insert. Using neomycin selection, apoA-IV-overexpressing (+AIV) and control (-AIV) clones were isolated for further study. Both undifferentiated (-D) and differentiated (+D) +AIV cells expressed 40- to 50-fold higher levels of apoA-IV mRNA and both intracellular and secreted apoA-IV protein compared with -AIV cells. Expression of other genes was not affected by apoA-IV overexpression in a manner that would contribute to enhanced lipid secretion. +D +AIV cells secreted 4.9-fold more labeled triacylglycerol (TG), 4.6-fold more labeled cholesteryl ester (CE), and 2-fold more labeled phospholipid (PL) as lipoproteins, mostly in the chylomicron/very low density lipoprotein (VLDL) density range. ApoA-IV overexpression in IPEC-1 cells enhances basolateral TG, CE, and PL secretion in chylomicron/VLDL particles. This enhancement is not associated with up-regulation of other genes involved in lipid transport. ApoA-IV may play a role in facilitating enterocyte lipid transport, particularly in the neonate receiving a diet of high fat breast milk.     

Genetic polymorphism of human plasma apolipoprotein A-IV is due to nucleotide substitutions in the apolipoprotein A-IV gene

Genetic polymorphism of human plasma apolipoprotein A-IV has been detected by isoelectric focusing techniques followed by immunoblotting. The molecular basis for this apoA-IV polymorphism has been elucidated. Analysis of the protein coding sequences of the apoA-IV alleles 1 and 2 revealed a single G to T substitution in the apoA-IV-2 allele. The point mutation, occurring in a region highly conserved among the mouse, rat, and human A-IV apolipoproteins, converts the glutamine at position 360 of the mature protein to a histidine. This amino acid substitution adds one positive charge unit to the apoA-IV-1 isoprotein (pI 4.97) thus creating the more basic apoA-IV-2 isoprotein (pI 5.02). Computer analysis of the apoA-IV-2 allele revealed that the single G to T substitution results in the loss of a BbvI and a Fnu4HI restriction enzyme site and in the formation of a new restriction site for the enzyme SfaNI. Protein primary and secondary structure predictions were largely unaffected by this amino acid exchange. These results on the structure of the apoA-IV-1 and apoA-IV-2 alleles suggest that the three other rare isoproteins (apoA-IV-0, apoA-IV-3, and apoA-IV-4) are also due to nucleotide and subsequent amino acid substitutions in the apoA-IV sequence.     

Cholesterol sensitivity of KIR2.1 depends on functional inter-links between the N and C termini

In recent years, cholesterol has been emerging as a major regulator of ion channel function. We have previously shown that cholesterol suppresses Kir2 channels, a subfamily of constitutively active strongly rectifying K⁺ channels. Furthermore, our earlier studies have shown that cholesterol sensitivity of Kir2 channels depends on a group of residues that form a belt-like structure around the cytosolic pore of the channel in proximity to the transmembrane domain. In this study, we focus on the contributions of different structural domains of Kir2 channels in the regulation of their cholesterol sensitivity. Focusing on the mildest mutation in the sensitivity belt, L222I, we show that the sensitivity of the channel to cholesterol can be restored by crosstalk between three distinct cytosolic regions: the C-terminal CD loop, the EF and GA loops of the C-terminus, and the βA sheet of the N-terminus. Thus, in addition to the importance of residues that affect the cytosolic G-loop gate in the sensitivity of Kir2 channels to cholesterol, our data suggest an important role to the interactions at the interface between the channel’s N- and C- termini that couple the intracellular domains of its four subunits during gating.     

Modulation of lipid synthesis,apolipoprotein biogenesis,and lipoprotein assembly by butyrate

Short-chain fatty acids (SCFAs) are potent modulators of the growth, function, and differentiation of intestinal epithelia. In addition, high-fiber diets may protect against the development of atherosclerosis because of their cholesterol-lowering effects due, in large part, to SCFA production, liver sterol metabolism, and bile acid excretion. Although the small gut plays a major role in dietary fat transport and contributes substantially to plasma cholesterol and lipoprotein homeostasis, the impact of SCFAs on intestinal lipid handling remains unknown. In the present study, the modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein secretion by butyrate was investigated in Caco-2 cells plated on permeable polycarbonate filters, which permit separate access to the upper and lower compartments of the monolayers. Highly differentiated and polarized cells (20 days of culture) were incubated for 20 h with 20 mM butyrate in the apical medium. In the presence of [14C]oleic acid, butyrate led to a significant reduction of secreted, labeled triglycerides (27%; P < 0.01) and phospholipids (25%; P < 0.05). Similarly, butyrate significantly decreased the incorporation of [14C]acetate into exported cholesteryl ester (49%; P < 0.005). As expected from these results, with [14C]oleic acid as a precursor, butyrate significantly (P < 0.05) diminished the delivery of radiolabeled chylomicrons and very low-density lipoproteins. In parallel, [35S]methionine pulse labeling of Caco-2 cells revealed the concomitant inhibitory effect of butyrate on the synthesis of apolipoproteins B-48 (28%; P < 0.05) and A-I (32%; P < 0.01). Collectively, our data indicate that butyrate may influence lipid metabolism in Caco-2 cells, thus suggesting a potential regulation of intestinal fat absorption and circulating lipoprotein concentrations.