👤 G S Shelness

Hepatic apolipoprotein A-IV (apoA-IV) expression is correlated with hepatic triglyceride (TG) content in mouse models of chronic hepatosteatosis, and steatosis-induced hepatic apoA-IV gene expression is regulated by nuclear transcription factor cAMP-responsive element-binding protein H (CREBH) processing. To define what aspects of TG homeostasis regulate hepatic CREBH processing and apoA-IV gene expression, several mouse models of attenuated VLDL particle assembly were subjected to acute hepatosteatosis induced by an overnight fast or short term ketogenic diet feeding. Compared with chow-fed C57BL/6 mice, fasted or ketogenic diet-fed mice displayed increased hepatic TG content, which was highly correlated (r Show less

Previous studies demonstrated that apolipoprotein A-IV (apoA-IV) promotes apoB lipoprotein-mediated triglyceride (TG) secretion in transfected enterocytes and hepatoma cells; however, evidence for a role in lipid transport in vivo is lacking. Using mouse models, we explored the role of apoA-IV in hepatic very low density lipoprotein-mediated lipid efflux under conditions that promote hepatic steatosis. Hepatic steatosis, induced by either high-fat diet or enhanced de novo lipogenesis caused by transgenic overexpression of SREBP-1a (SREBP-1a(Tg)), was associated with up to a 43-fold induction of hepatic apoA-IV mRNA and protein levels. In both models, a positive linear correlation between hepatic TG content and apoA-IV mRNA abundance was observed (r(2)=0.8965). To examine whether induction of apoA-IV affected hepatic TG secretion, SREBP-1a(Tg) mice were crossed with Apoa4 knockout mice. With Triton blockade of peripheral lipolysis, SREBP-1a(Tg)/Apoa4 knockout mice demonstrated a 24% reduction in hepatic TG secretion rate, relative to SREBP-1a(Tg) controls, but no change in apoB production. Negative stain electron microscopy revealed a 33% decrease in the abundance of secreted very low density lipoprotein particles with diameters ≥ 120 nm. Conversely, mice infected with a recombinant human apoA-IV adenovirus demonstrated a 52% increase in the hepatic TG secretion rate, relative to controls, a 38% reduction in liver TG content, and a 43% increase in large diameter (≥ 120 nm) very low density lipoprotein particles, with no change in apoB secretion. Hepatic steatosis in mice induces hepatic apoA-IV expression, which in turn promotes lipoprotein particle expansion and reduces hepatic lipid burden without increasing the number of secreted atherogenic apoB-containing lipoprotein particles. Show less

Although the evidence linking apoA-IV expression and triglyceride (TG)-rich lipoprotein assembly and secretion is compelling, the intracellular mechanisms by which apoA-IV could modulate these processes remain poorly understood. We therefore examined the functional impact of apoA-IV expression on endogenous apoB, TG, and VLDL secretion in stably transfected McA-RH7777 rat hepatoma cells. Expression of apoA-IV modified with the endoplasmic reticulum (ER) retention signal KDEL (apoA-IV-KDEL) dramatically decreased both the rate and efficiency of endogenous apoB secretion, suggesting a presecretory interaction between apoA-IV-KDEL and apoB or apoB-containing lipoproteins. Expression of native apoA-IV using either a constitutive or tetracycline-inducible promoter delayed the initial rate of apoB secretion and reduced the final secretion efficiency by ∼40%. However, whereas apoA-IV-KDEL reduced TG secretion by 75%, expression of native apoA-IV caused a 20-35% increase in TG secretion, accompanied by a ∼55% increase in VLDL-associated apoB, an increase in the TG:phospholipid ratio of secreted d < 1.006 lipoproteins, and a 10.1 nm increase in peak VLDL(1) particle diameter. Native apoA-IV expression had a negligible impact on expression of the MTP gene. These data suggest that by interacting with apoB in the secretory pathway, apoA-IV alters the trafficking kinetics of apoB-containing TG-rich lipoproteins through cellular lipidation compartments, which in turn, enhances particle expansion and increases TG secretion. Show less

Apolipoprotein A-V (apoA-V) is a potent regulator of intravascular triglyceride (TG) metabolism, yet its plasma concentration is very low compared with that of other apolipoproteins. To examine the basis for its low plasma concentration, the secretion efficiency of apoA-V was measured in stably transfected McA-RH7777 rat hepatoma cells. Pulse-chase experiments revealed that only ∼20% of newly synthesized apoA-V is secreted into culture medium within 3 h postsynthesis and that ∼65% undergoes presecretory turnover; similar results were obtained with transfected nonhepatic Chinese hamster ovary cells. ApoA-V secreted by McA-RH7777 cells was not associated with cell surface heparin-competable binding sites. When stably transfected McA-RH7777 cells were treated with oleic acid, the resulting increase in TG synthesis caused a reduction in apoA-V secretion, a reciprocal increase in cell-associated apoA-V, and movement of apoA-V onto cytosolic lipid droplets. In a stably transfected doxycycline-inducible McA-RH7777 cell line, apoA-V expression inhibited TG secretion by ∼50%, increased cellular TG, and reduced Z-average VLDL(1) particle diameter from 81 to 67 nm; however, no impact on apoB secretion was observed. These data demonstrate that apoA-V inefficiently traffics within the secretory pathway, that its intracellular itinerary can be regulated by changes in cellular TG accumulation, and that apoA-V synthesis can modulate VLDL TG mobilization and secretion. Show less

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans. Show less

To examine the role of apolipoprotein A-IV (apoA-IV) in the intracellular trafficking and secretion of apoB, COS cells were cotransfected with microsomal triglyceride transfer protein (MTP), apoB-41 (amino terminal 41% of apoB), and either native apoA-IV or apoA-IV modified with the carboxy-terminal endoplasmic reticulum (ER) retention signal, KDEL (apoA-IV-KDEL). As expected, apoA-IV-KDEL was inefficiently secreted relative to native apoA-IV. Coexpression of apoB-41 with apoA-IV-KDEL reduced the secretion of apoB-41 by approximately 80%. The apoA-IV-KDEL effect was specific, as neither KDEL-modified forms of human serum albumin or apoA-I affected apoB-41 secretion. Similar results were observed in McA-RH7777 rat hepatoma cells, which express endogenous MTP. The full inhibitory effect of apoA-IV-KDEL on apoB secretion was observed only for forms of apoB containing a minimum of the amino-terminal 25% of the protein (apoB-25). However, apoA-IV-KDEL inhibited the secretion of both lipid-associated and lipid-poor forms of apoB-25. Dual-label immunofluorescence microscopy of cells transfected with native apoA-IV and apoB-25 revealed that both apolipoproteins were localized to the ER and Golgi, as expected. However, when apoA-IV-KDEL was cotransfected with apoB-25, both proteins localized primarily to the ER. These data suggest that apoA-IV may physically interact with apoB in the secretory pathway, perhaps reflecting a role in modulating the process of triglyceride-rich lipoprotein assembly and secretion. Show less

Apolipoprotein A-V (apoA-V), the newest member of the plasma apolipoprotein family, was recently discovered by comparison of the mouse and human genomes. Studies in rodents and population surveys of human apoA-V polymorphisms have noted a strong effect of apoA-V on plasma triglyceride levels. Toward the elucidation of the biologic function of apoA-V, we used spectroscopic and surface chemistry techniques to probe its structure and interfacial activity. Computer-assisted sequence analysis of apoA-V predicts that it is very hydrophobic, contains a significant amount of alpha-helical secondary structure, and probably is composed of discrete structural regions with varying degrees of lipid affinity. Fluorescence spectroscopy of recombinant human apoA-V provided evidence of tertiary folding, and light scattering studies indicated that apoA-V transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes with an efficiency similar to that of apoA-I. Surface chemistry techniques revealed that apoA-V displays high affinity, low elasticity, and slow binding kinetics at hydrophobic interfaces, properties we propose may retard triglyceride-rich particle assembly. Metabolic labeling and immunofluorescence studies of COS-1 cells transfected with human apoA-V demonstrated that apoA-V is poorly secreted, remains associated with the endoplasmic reticulum, and does not traffic to the Golgi. Given that overexpression of the apoA-V gene lowers plasma triglycerides in mice, these data together suggest that apoA-V may function intracellularly to modulate hepatic VLDL synthesis and/or secretion. Show less

Viscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/water interface apolipoproteins A-IV and B-17 displayed wide area - tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 3 10(-)(3) sec(-)(1), whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 3 10(-)(3) sec(-)(1). Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure. We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles. Show less