The intracellular transport of chylomicrons requires the small GTPase, Sar1b

PURPOSE OF REVIEW: The transport of lipoproteins through the secretory pathways of enterocytes and hepatocytes is pivotal for whole-body lipid homeostasis. This review focuses on the assembly and structural evolution of COPII (coat protein) transport carriers that are essential for the transport of chylomicrons from the endoplasmic reticulum to the Golgi apparatus. RECENT FINDINGS: The assembly of endoplasmic reticulum to Golgi transport carriers commences with the coating of specific areas of the endoplasmic reticulum membrane with Sar1-GTP and the Sec23/24 heterodimer. An important advance has been the crystallographic analysis of the Sar1-Sec23/24 complex. The proteins form a bow-tie shaped structure, with a concave face that seems to match the curvature of transport carriers. Mammalian cells produce two isoforms of Sar1, designated Sar1a and Sar1b, both of which are expressed in enterocytes. Sar1b is defective in chylomicron retention disease and Anderson disease, two rare recessive disorders characterized by severe fat malabsorption and a failure to thrive in infancy. Patients with chylomicron retention disease and Anderson disease selectively retain chylomicron-like particles within membrane-bound compartments. By analogy with procollagen, chylomicrons may drive the formation of endoplasmic reticulum to Golgi transport carriers from endoplasmic reticulum sites close to, but separate from, domains of the endoplasmic reticulum coated with Sar1-Sec23/24. The COPII machinery also mediates the transport of VLDL to the Golgi. SUMMARY: New insights into the role of the COPII machinery in the intracellular transport of cargo, including chylomicrons and VLDL, may suggest new drug targets for ameliorating the lipid abnormalities of the metabolic syndrome.     

Proteomic profiling of intestinal prechylomicron transport vesicle (PCTV)-associated proteins in an animal model of insulin resistance (94 char)

Intestinal overproduction of apolipoprotein B (apoB)-48-containing chylomicrons is increasingly recognized as an underlying factor in metabolic dyslipidemia commonly observed in insulin-resistant states. Enhanced chylomicron assembly and secretion has been documented in animal models of insulin resistance, but the underlying mechanistic factors are unknown. Chylomicron assembly occurs through a series of complex vesicular interactions involving prechylomicron transport vesicles (PCTVs), which transport lipids from the endoplasmic reticulum (ER) to the Golgi. We report proteomic profiles of PCTVs isolated from the enteric ER in the small intestine of the fructose-fed hamster, an established model of diet-induced insulin resistance. Using 2D gel electrophoresis and tandem mass spectrometry, PCTVs were characterized and proteomic profiles of PCTV-associated proteins from insulin-resistant and control enterocytes were developed, with the intention of identifying proteins involved in insulin signaling attenuation and lipoprotein overproduction. A number of PCTV-associated proteins were found to be differentially expressed including microsomal triglyceride transfer protein (MTP), apoB-48, Sar1 and VAMP7. We postulate that altered expression of Sar1 and MTP may contribute to increased chylomicron assembly in the fructose-fed hamster. These findings have increased our understanding of the intracellular assembly and transport of nascent chylomicrons and potential cellular factors responsible for lipoprotein overproduction in insulin-resistant states.     

Ezetimibe impairs transcellular lipid trafficking and induces large lipid droplet formation in intestinal absorptive epithelial cells

Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1) protein, which mediates intracellular cholesterol trafficking from the brush border membrane to the endoplasmic reticulum, where chylomicron assembly takes place in enterocytes or in the intestinal absorptive epithelial cells. Cholesterol is a minor lipid constituent of chylomicrons; however, whether or not a shortage of cholesterol attenuates chylomicron assembly is unknown. The aim of this study was to examine the effect of ezetimibe, a potent NPC1L1 inhibitor, on trans-epithelial lipid transport, and chylomicron assembly and secretion in enterocytes. Caco-2 cells, an absorptive epithelial model, grown onto culture inserts were given lipid micelles from the apical side, and chylomicron-like triacylglycerol-rich lipoprotein secreted basolaterally were analyzed after a 24-h incubation period in the presence of ezetimibe up to 50 μM. The secretion of lipoprotein and apolipoprotein B48 were reduced by adding ezetimibe (30% and 34%, respectively). Although ezetimibe allowed the cells to take up cholesterol normally, the esterification was abolished. Meanwhile, oleic acid esterification was unaffected. Moreover, ezetimibe activated sterol regulatory element-binding protein 2 by approximately 1.5-fold. These results suggest that ezetimibe limited cellular cholesterol mobilization required for lipoprotein assembly. In such conditions, large lipid droplet formation in Caco-2 cells and the enterocytes of mice were induced, implying that unprocessed triacylglycerol was sheltered in these compartments. Although ezetimibe did not reduce the post-prandial lipid surge appreciably in triolein-infused mice, the results of the present study indicated that pharmacological actions of ezetimibe may participate in a novel regulatory mechanism for the efficient chylomicron assembly and secretion.     

Intestine-specific MTP and global ACAT2 deficiency lowers acute cholesterol absorption with chylomicrons and HDLs

Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.     

Gut triglyceride production

Our knowledge of how the body absorbs triacylglycerols (TAG) from the diet and how this process is regulated has increased at a rapid rate in recent years. Dietary TAG are hydrolyzed in the intestinal lumen to free fatty acids (FFA) and monoacylglycerols (MAG), which are taken up by enterocytes from their apical side, transported to the endoplasmic reticulum (ER) and resynthesized into TAG. TAG are assembled into chylomicrons (CM) in the ER, transported to the Golgi via pre-chylomicron transport vesicles and secreted towards the basolateral side. In this review, we mainly focus on the roles of key proteins involved in uptake and intracellular transport of fatty acids, their conversion to TAG and packaging into CM. We will also discuss intracellular transport and secretion of CM. Moreover, we will bring to light few factors that regulate gut triglyceride production. Furthermore, we briefly summarize pathways involved in cholesterol absorption. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Gut triglyceride production

Our knowledge of how the body absorbs triacylglycerols (TAG) from the diet and how this process is regulated has increased at a rapid rate in recent years. Dietary TAG are hydrolyzed in the intestinal lumen to free fatty acids (FFA) and monoacylglycerols (MAG), which are taken up by enterocytes from their apical side, transported to the endoplasmic reticulum (ER) and resynthesized into TAG. TAG are assembled into chylomicrons (CM) in the ER, transported to the Golgi via pre-chylomicron transport vesicles and secreted towards the basolateral side. In this review, we mainly focus on the roles of key proteins involved in uptake and intracellular transport of fatty acids, their conversion to TAG and packaging into CM. We will also discuss intracellular transport and secretion of CM. Moreover, we will bring to light few factors that regulate gut triglyceride production. Furthermore, we briefly summarize pathways involved in cholesterol absorption. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Mechanisms involved in vitamin E transport by primary enterocytes and in vivo absorption

It is generally believed that vitamin E is absorbed along with chylomicrons. However, we previously reported that human colon carcinoma Caco-2 cells use dual pathways, apolipoprotein B (apoB)-lipoproteins and HDLs, to transport vitamin E. Here, we used primary enterocytes and rodents to identify in vivo vitamin E absorption pathways. Uptake of [(3)H]alpha-tocopherol by primary rat and mouse enterocytes increased with time and reached a maximum at 1 h. In the absence of exogenous lipid supply, these cells secreted vitamin E with HDL. Lipids induced the secretion of vitamin E with intermediate density lipoproteins, and enterocytes supplemented with lipids and oleic acid secreted vitamin E with chylomicrons. The secretion of vitamin E with HDL was not affected by lipid supply but was enhanced when incubated with HDL. Microsomal triglyceride transfer protein inhibition reduced vitamin E secretion with chylomicrons without affecting its secretion with HDL. Enterocytes from Mttp-deficient mice also secreted less vitamin E with chylomicrons. In vivo absorption of [(3)H]alpha-tocopherol by mice after poloxamer 407 injection to inhibit lipoprotein lipase revealed that vitamin E was associated with triglyceride-rich lipoproteins and small HDLs containing apoB-48 and apoA-I. These studies indicate that enterocytes use two pathways for vitamin E absorption. Absorption with chylomicrons is the major pathway of vitamin E absorption. The HDL pathway may be important when chylomicron assembly is defective and can be exploited to deliver vitamin E without increasing fat consumption.     

The 1991 Borden Award Lecture. Selected aspects of intraluminal and intracellular phases of intestinal fat absorption.

The recognition of chylomicrons as dietary lipid transporters dates back to more than 70 years and marks a milestone in lipoprotein history. Conventionally, three phases constitute the process of absorption of exogenous fat: intraluminal, intestinal, and delivery. The intraluminal phase includes chemical hydrolysis by lipolytic enzymes and the micellar solubilization of lipolytic products by bile acids. The intestinal phase comprises the diffusion of micelles through the unstirred water layer, passive diffusion across the microvillous membrane of the enterocyte, and the formation of lipid-carrying lipoproteins. The delivery phase involves the exocytosis of chylomicrons from the absorptive cells and their subsequent removal by lymphatic structures and the systemic circulation. The precise steps and factors involved in all phases of chylomicron synthesis are not yet known, but both experimental and clinical studies have been helpful. Of the inborn metabolic disorders, the prerequisite function of apolipoprotein (apo B) for the assembly and release of lipoprotein particles stood out. Moreover, evidence emerged that the enterocyte produces apo B-100 in addition to apo B-48. Calcium and essential fatty acid status originates as determinants for triglyceride-rich particle synthesis. Furthermore, the developmental changes and regulatory factors of lipoprotein elaboration represent excellent tools in the study of the intracellular mechanisms of lipid transport.     

Transport of vitamin E by differentiated Caco-2 cells

In hepatocytes, vitamin E is secreted via the efflux pathway and is believed to associate with apolipoprotein B (apoB)-lipoproteins extracellularly. The molecular mechanisms involved in the uptake, intracellular trafficking, and secretion of dietary vitamin E by the intestinal cells are unknown. We observed that low concentrations of Tween-40 were better for the solubilization and delivery of vitamin E to differentiated Caco-2 cells, whereas high concentrations of Tween-40 and sera inhibited this uptake. Vitamin E uptake was initially rapid and then reached saturation. Subcellular localization revealed that vitamin E primarily accumulated in microsomal membranes. Oleic acid (OA) treatment, which induces chylomicron assembly and secretion, decreased microsomal membrane-bound vitamin E in a time-dependent manner. To study secretion, differentiated Caco-2 cells were pulse-labeled with vitamin E and chased in the presence and absence of OA. In the absence of OA, vitamin E was associated with intestinal high density lipoprotein (I-HDL), whereas OA-treated cells secreted vitamin E with I-HDL and chylomicrons. No extracellular transfer of vitamin E between these lipoproteins was observed. Glyburide, an antagonist of ABCA1, partially inhibited its secretion with I-HDL, whereas plasma HDL increased vitamin E efflux. An antagonist of microsomal triglyceride transfer protein, brefeldin A, and monensin specifically inhibited vitamin E secretion with chylomicrons. These studies indicate that vitamin E taken up by Caco-2 cells is stored in the microsomal membranes and secreted with chylomicrons and I-HDL. Transport via I-HDL might contribute to vitamin E absorption in patients with abetalipoproteinemia receiving large oral doses of the vitamin.     

Multiple,independently regulated pathways of cholesterol transport across the intestinal epithelial cells

The present study provides a new understanding about the mechanisms involved in cholesterol absorption by the intestinal cells. Contrary to general belief, our data show that newly absorbed cholesterol is neither immediately available for secretion with apoB lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated, apoB-dependent and -independent, pathways. Cholesterol secretion by the apoB-dependent pathway is induced by oleic acid, is repressed by microsomal triglyceride transfer protein inhibitors, and occurs only with larger apoB-containing lipoproteins. ApoB-independent pathways do not require microsomal triglyceride transfer protein and involve efflux mediated by ABCA1, high density lipoprotein assembly, and possibly other unknown mechanisms. There are at least two different metabolic pools of cholesterol. The newly absorbed and pre-absorbed cholesterol are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. In contrast to compartmentalization for secretion, these two metabolic pools are equally accessible for cellular esterification. The esterified cholesterol is mainly secreted by the apoB-dependent pathway, whereas both the pathways are involved in the secretion of free cholesterol. Thus, enterocytes transport exogenous cholesterol by several independently regulated pathways raising the possibility that targeting of apoB-independent pathways may result in selective inhibition of cholesterol transport without affecting triglyceride transport.     

Carboxylesterase1/Esterase-x Regulates Chylomicron Production in Mice

Elevated postprandial plasma triacylglycerol (TG) concentrations are commonly associated with obesity and the risk of cardiovascular disease. Dietary fat contributes to this condition through the production of chylomicrons. Carboxylesterases have been mainly studied for their role in drug metabolism, but recently they have been shown to participate in lipid metabolism; however, their role in intestinal lipid metabolism is unknown. Carboxylesterase1/esterase-x (Ces1/Es-x) deficient mice become obese, hyperlipidemic and develop hepatic steatosis even on standard chow diet. Here, we aimed to explore the role of Ces1/Es-x in intestinal lipid metabolism. Six-month old wild-type and Ces1/Es-x deficient mice were maintained on chow diet and intestinal lipid metabolism and plasma chylomicron clearance were analyzed. Along the intestine Ces1/Es-x protein is expressed only in proximal jejunum. Ablation of Ces1/Es-x expression results in postprandial hyperlipidemia due to increased secretion of chylomicrons. The secreted chylomicrons have aberrant protein composition, which results in their reduced clearance. In conclusion, Ces1/Es-x participates in the regulation of chylomicron assembly and secretion. Ces1/Es-x might act as a lipid sensor in enterocytes regulating chylomicron secretion rate. Ces1/Es-x might represent an attractive pharmacological target for the treatment of lipid abnormalities associated with obesity, insulin resistance and fatty liver disease.     

Is the Golgi apparatus the obligatory final step for lipoprotein secretion by intestinal cells?

It is currently admitted that the synthesis and excretion of triglyceride-rich lipoproteins (chylomicrons and 'small chylomicrons') by intestinal epithelial cells involves the Golgi apparatus as an obligatory final step before exocytosis. The cells of the proximal intestine of the trout are an excellent model for investigating functional compartmentalization in the course of lipid absorption. Using this model, our data invalidate morphological data which were the basis for considering the Golgi apparatus as the mandatory final stage for their secretion. In particular, we show that triglyceride-rich particles can be transported directly from the endoplasmic reticulum to the intercellular space. Two pathways of intestinal lipoprotein excretion appear to coexist. One follows the classical export route, the second functions in a manner that bypasses the Golgi apparatus. The arguments used to affirm the requirement for the Golgi apparatus as a final step (glycosylation of apoprotein B, membrane vehicle for exocytosis) are discussed.     

Distribution of apolipoproteins A-I and B among intestinal lipoproteins

Chylomicrons and very low density lipoproteins (VLDL) are produced by the intestine and these nascent particles are thought to be similar to their counterparts in intestinal lymph. To study the relationship between these lipoproteins within the cell and those secreted into the lamina propria and lymph, we have isolated enterocytes, lamina propria, and mesenteric lymph from rats while fasted and after corn oil feeding. Apolipoprotein A-I and B content were measured by radioimmunoassay in cell, lamina propria, and lymph fractions separated by Sepharose 6B and 10% agarose chromatography, and by KBr isopycnic density centrifugation. ApoA-I in the cell and the underlying lamina propria was found partly in those fractions in which chylomicron and very low density lipoproteins (chylo-VLDL) and high density lipoproteins (HDL) elute, but more abundantly where unassociated 125I-labeled apoA-I was eluted. In the lymph, however, 74% of apoA-I eluted in the HDL region and no peak of free apoA-I was found. ApoB and apoC-III within the enterocyte were found distributed in the position of particles eluting not only with chylomicrons and VLDL, but also in the regions corresponding to LDL and HDL. In the lamina propria and lymph, on the other hand, most of the apoB was found in the region of VLDL and chylomicrons. These results indicate that the patterns in lymph lipoproteins and the lamina propria do not exactly mirror the distribution of apoA-I and B among lipoproteins inside the cell. This may be because intracellular apoproteins may be unassociated with lipoproteins, or they could be associated with lipoproteins in various stages of assembly of protein with lipids. Furthermore, the apoprotein composition of intestinal lipoproteins is altered after secretion from the enterocyte. Finally, not all apoproteins seem to be secreted in association with identifiable lipoprotein particles from the enterocyte.     

Intracellular apoA-I and apoB distribution in rat intestine is altered by lipid feeding

Intracellular forms of chylomicrons, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) have previously been isolated from the rat intestine. These intracellular particles are likely to be nascent precursors of secreted lipoproteins. To study the distribution of intracellular apolipoprotein among nascent lipoproteins, a method to isolate intracellular lipoproteins was developed and validated. The method consists of suspending isolated enterocytes in hypotonic buffer containing a lipase inhibitor, rupturing cell membranes by nitrogen cavitation, and isolating lipoproteins by sequential ultracentrifugation. ApoB and apoA-I mass are determined by radioimmunoassay and newly synthesized apolipoprotein characterized following [3H]leucine intraduodenal infusion. Intracellular chylomicron, VLDL, low density lipoprotein (LDL), and HDL fractions were isolated and found to contain apoB, and apoA-IV, and apoA-I. In the fasted animal, less than 10% of total intracellular apoB and apoA-I was bound to lipoproteins and 7% of apoB and 35% of apoA-I was contained in the d 1.21 g/ml infranatant. The remainder of intracellular apolipoprotein was in the pellets of centrifugation. Lipid feeding doubled the percentage of intracellular apoA-I bound to lipoproteins and increased the percentage of intracellular apoB bound to lipoproteins by 65%. Following lipid feeding, the most significant increase was in the chylomicron apoB and HDL apoA-I fractions. These data suggest that in the fasting state, 90% of intracellular apoB and apoA-I is not bound to lipoproteins. Lipid feeding shifts intracellular apolipoprotein onto lipoproteins, but most intracellular apolipoprotein remains non-lipoprotein bound. The constant presence of a large non-lipoprotein-bound pool suggests that apolipoprotein synthesis is not the rate limiting step in lipoprotein assembly or secretion.     

Further studies on the mechanism of inhibition of intestinal chylomicron transport by Pluronic L-81

This study explored further the hypothesis that intestinal cells have two pathways for producing large triacylglycerol-rich lipoprotein particles. The hydrophobic surfactant Pluronic L-81 (L-81) inhibits formation of chylomicrons (containing triacylglycerol synthesized from dietary fatty acids and monoacylglycerol, through the monoacylglycerol pathway), but not formation of very-low-density lipoproteins. L-81 does not inhibit lymphatic lipid transport during infusion of egg phosphatidylcholine, whose fatty acid is processed through the alpha-glycerol phosphate pathway and is transported in lymph in very-low-density lipoproteins. Thus, the first part of this study tested whether L-81 cannot inhibit the alpha-glycerol phosphate pathway, and thus L-81 can only affect chylomicron lipid secretion. Intestinal lymph fistula rats were infused with a lipid emulsion containing [1-14C]oleic acid, but no monoacylglycerol, to ensure that the oleic acid will be channeled to the alpha-glycerol phosphate pathway. Experimental rats received 1 mg/h of L-81 in their emulsion whereas control rats lacked L-81. Lymphatic triacylglycerol output, measured both chemically and radioactively, was markedly suppressed in the experimental rats as compared to the controls. Thus, these data indicate that the reason why lipid transport was unaffected by L-81 when egg phosphatidylcholine was infused was not because of the pathway used for the resynthesis of triacylglycerol from phosphatidylcholine. In the second part of this study, we measured the appearance time for chylomicron (in control rats) and for very-low-density lipoprotein (in L-81-treated rats). The appearance time is defined as the time between placement of radioactive fatty acid into the intestinal lumen and the appearance of radioactive lipid in the central lacteal. The average appearance time for the control rats was 10.8 min, which was significantly shorter than the 16.2 min in the L-81-treated experimental rats. This difference in appearance time further supports the hypothesis that chylomicron and very-low-density lipoprotein are packaged separately in the enterocytes and only the formation of chylomicron is inhibited by L-81.     

Chylomicrons promote intestinal absorption of lipopolysaccharides

Recent data suggest that dietary fat promotes intestinal absorption of lipopolysaccharides (LPS) from the gut microflora, which might contribute to various inflammatory disorders. The mechanism of fat-induced LPS absorption is unclear, however. Intestinal-epithelial cells can internalize LPS from the apical surface and transport LPS to the Golgi. The Golgi complex also contains newly formed chylomicrons, the lipoproteins that transport dietary long-chain fat through mesenteric lymph and blood. Because LPS has affinity for chylomicrons, we hypothesized that chylomicron formation promotes LPS absorption. In agreement with our hypothesis, we found that CaCo-2 cells released more cell-associated LPS after incubation with oleic-acid (OA), a long-chain fatty acid that induces chylomicron formation, than with butyric acid (BA), a short-chain fatty acid that does not induce chylomicron formation. Moreover, the effect of OA was blocked by the inhibitor of chylomicron formation, Pluronic L-81. We also observed that intragastric triolein (TO) gavage was followed by increased plasma LPS, whereas gavage with tributyrin (TB), or TO plus Pluronic L-81, was not. Most intestinally absorbed LPS was present on chylomicron remnants (CM-R) in the blood. Chylomicron formation also promoted transport of LPS through mesenteric lymph nodes (MLN) and the production of TNFalpha mRNA in the MLN. Together, our data suggest that intestinal epithelial cells may release LPS on chylomicrons from cell-associated pools. Chylomicron-associated LPS may contribute to postprandial inflammatory responses or chronic diet-induced inflammation in chylomicron target tissues.     

Pulmonary surfactant phosphatidylcholine transport bypasses the brefeldin A sensitive compartment of alveolar type II cells

Brefeldin A (BFA) causes disassembly of the Golgi apparatus and blocks protein transport to this organelle from the endoplasmic reticulum. However, there still remains considerable ambiguity regarding the involvement of the Golgi apparatus in glycerolipid transport pathways. We examined the effects of BFA upon the intracellular translocation of phosphatidylcholine in alveolar type II cells, that synthesize, transport, store and secrete large amounts of phospholipid for regulated exocytosis. BFA at concentrations as high as 10 microg/ml failed to alter the assembly of phosphatidylcholine into lamellar bodies, the specialized storage organelles for pulmonary surfactant. The same concentration of BFA was also ineffective at altering the secretion of newly synthesized phosphatidylcholine from alveolar type II cells. In contrast, concentrations of the drug of 2.5 microg/ml completely arrested newly synthesized lysozyme secretion from the same cells, indicating that BFA readily blocked protein transport processes in alveolar type II cells. The disassembly of the Golgi apparatus in alveolar type II cells following BFA treatment was also demonstrated by showing the redistribution of the resident Golgi protein MG-160 to the endoplasmic reticulum. These results indicate that intracellular transport of phosphatidylcholine along the secretory pathway in alveolar type II cells proceeds via a BFA insensitive route and does not require a functional Golgi apparatus.     

Pluronic L81 affects the lipid particle sizes and apolipoprotein B conformation

The chylomicron assembly has been proposed to involve the core expansion of apolipoprotein B (apoB)-containing primordial lipoproteins by fusing with triglyceride-rich lipid droplets. We examined the effects of an inhibitor of chylomicron secretion, Pluronic L81, on triolein-phosphatidylcholine emulsions and low density lipoproteins (LDL) which were used for the models of lipid droplets and primordial lipoproteins, respectively. We showed by dynamic light scattering that the sizes of lipid emulsions and LDL were increased in the presence of Pluronic L81. The binding of apoB-100 to lipid emulsions was enhanced by Pluronic L81. CD and fluorescence lifetime measurements revealed that Pluronic L81 altered the secondary structure of apoB-100 with an increased local hydration. The proper hydrophilic-to-hydrophobic balance of Pluronic L81 is important for these actions. It is proposed that Pluronic L81 inhibits the secretion of chylomicrons by leading the excess core expansion of the primordial lipoproteins and the conformational modification of apoB.     

Intracellular transport of endocytosed chylomicron [3H]retinyl ester in rat liver parenchymal cells. Evidence for translocation of a [3H]retinoid from endosomes to endoplasmic reticulum

The intracellular transport of chylomicron remnants labeled with [3H]retinyl ester was studied in rat liver parenchymal cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. The data presented indicate that endocytosed chylomicron remnant [3H]retinyl ester initially is located in low density endosomes. Radioactivity is subsequently transferred to a denser vesicle. Equilibrium as well as rate zonal centrifugation suggest that this denser [3H] retinoid-containing vesicle may represent endoplasmic reticulum. We have compared the intracellular transport of chylomicron remnant [3H]retinyl ester and 125I-asialofetuin. The receptor-mediated endocytosis of asialoglycoproteins in rat liver parenchymal cells is a thoroughly studied system. Our results suggest that the [3H] retinoid and 125I-asialofetuin follow the same path initially to the endosomes. After transit in endosomes, the intracellular transport differs. While asialofetuin is transported to the lysosomes, the retinoid is probably transferred to the endoplasmic reticulum.