Inhibition of chylomicron assembly leads to dissociation of hepatic steatosis from inflammation and fibrosis.

Regulating dietary fat absorption may impact progression of nonalcoholic fatty liver disease (NAFLD). Here, we asked if inducible inhibition of chylomicron assembly, as observed in intestine-specific microsomal triglyceride (TG) transfer protein knockout mice (Mttp-IKO), could retard NAFLD progression and/or reverse established fibrosis in two dietary models. Mttp-IKO mice fed a methionine/choline-deficient (MCD) diet exhibited reduced hepatic TGs, inflammation, and fibrosis, associated with reduced oxidative stress and downstream activation of c-Jun N-terminal kinase and nuclear factor kappa B signaling pathways. However, when Mttpflox mice were fed an MCD for 5 weeks and then administered tamoxifen to induce Mttp-IKO, hepatic TG was reduced, but inflammation and fibrosis were increased after 10 days of reversal along with adaptive changes in hepatic lipogenic mRNAs. Extending the reversal time, following 5 weeks of MCD feeding to 30 days led to sustained reductions in hepatic TG, but neither inflammation nor fibrosis was decreased, and both intestinal permeability and hepatic lipogenesis were increased. In a second model, similar reductions in hepatic TG were observed when mice were fed a high-fat/high-fructose/high-cholesterol (HFFC) diet for 10 weeks, then switched to chow ± tamoxifen (HFFC → chow) or (HFFC → Mttp-IKO chow), but again neither inflammation nor fibrosis was affected. In conclusion, we found that blocking chylomicron assembly attenuates MCD-induced NAFLD progression by reducing steatosis, oxidative stress, and inflammation. In contrast, blocking chylomicron assembly in the setting of established hepatic steatosis and fibrosis caused increased intestinal permeability and compensatory shifts in hepatic lipogenesis that mitigate resolution of inflammation and fibrogenic signaling despite 50-90-fold reductions in hepatic TG.

Intestinal lymphatic transport of halofantrine in rats assessed using a chylomicron flow blocking approach: the influence of polysorbate 60 and 80.

The aim of the study was to examine the effects of polysorbate 60 and 80 on intestinal lymphatic transport of a poorly water-soluble compound, halofantrine, using a chylomicron flow blocking approach in rats. Male Sprague-Dawley rats were pretreated intraperitoneally with 3.0 mg/kg cycloheximide or saline. One hour later, rats were dosed with 6.7 mg/kg halofantrine in 0.4 or 1.0 g/kg polysorbate 60 or 80, 0.33 g/kg soybean oil or 0.33 g/kg soybean oil+1.0 g/kg polysorbate 80 by gavage, and plasma samples were collected. The fraction of halofantrine transported to the lymphatic system was determined as the difference between the bioavailability in saline-pretreated rats and cycloheximide-pretreated rats. No significant differences in halofantrine transport to the systemic blood and in the deduced lymphatic transport were observed between the two polysorbates, at the tested dosages. The lymphatic transport of halofantrine was the same whether dosing with polysorbate 60, polysorbate 80 or soybean oil; accordingly both surfactants can be used as lymphotropic carriers. Furthermore, there was a good correlation between the halofantrine transport to blood and lymphatics in the chylomicron flow blocking model and published results from the mesenteric lymph-cannulated model.

Evaluation of a chylomicron flow blocking approach to investigate the intestinal lymphatic transport of lipophilic drugs.

The purpose of this study was to examine the feasibility of investigating the lymphatic transport of drugs in vivo utilizing known chylomicron flow blocking substances. Vitamin D(3) (0.5 mg/kg), a model lipophilic molecule, was administered to rats with blocked chylomicron flow, induced by either cycloheximide injection (3 mg/kg), colchicine injection (5 mg/kg) or intraduodenal infusion of pluronic L-81 (1 mg/h). The effect of these experimental models on the absorption of Vitamin D(3) was compared to the outcomes of the mesenteric lymph duct cannulated rat model. The oral d-xylose loading test was used to verify that other intestinal absorptive functions were not affected. Colchicine treatment induced severe adverse effects whereas pluronic L-81 and the cycloheximide models did not affect other absorption pathways and did not cause apparent adverse effects. Vitamin D(3) absorption in these two models was in good correlation to the mesenteric lymph duct cannulation model (25% non-lymphatic relative bioavailability) indicating that the incorporation of the lipophilic molecule into the chylomicron is an essential step in the cascade of lymphatic absorption. Moreover, the data suggest that the drug association with the chylomicron occurs at an early stage of its assembly process. The results also specify that lymphatic absorption and portal blood absorption are separate pathways that are not affected by each other. In conclusion, the chemical blockage of chylomicron flow provides a potential approach for lymphatic transport investigation, and may elucidate processes involving in the absorption of lipophilic compounds.

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.

Effect of lipoprotein lipase on binding of chylomicrons to LDL receptor-deficient Chinese hamster ovary cells.

The authors investigated the binding of human plasma 125I-labelled chylomicrons to Chinese hamster ovary (CHO) cells, i.e. native CHO cells are mutant ldl-A7 cells lacking the low-density lipoproteins receptor, in the absence and presence of exogenous bovine milk lipoprotein lipase (LPL) in the culture medium. Only a small amount of binding to either cell was observed in the absence of added LPL. Exogenously added LPL increased the specific binding of chylomicrons to ldl-A7 cells, as well as to native CHO cells. The enhanced binding of chylomicrons to ldl-A7 cells or native CHO cells by LPL was inhibited by heparinase and a monoclonal antibody against LPL (5D2) which recognizes the carboxyl terminal of LPL. However, the enhanced binding was not inhibited by 1 M NaCl, which abolishes the enzymatic activity of LPL in either ldl-A7 cell or native CHO cells. These results suggest that LPL enhances the binding of chylomicrons to heparan sulphate proteoglycans of CHO cells, and that it is the carboxyl terminal of LPL but not the enzymatic activity of LPL that is essential for LPL to mediate the binding of chylomicrons to CHO cells.

Retinyl ester secretion by intestinal cells: a specific and regulated process dependent on assembly and secretion of chylomicrons.

Retinyl esters (RE) have been used extensively as markers to study chylomicron (CM) catabolism because they are secreted in the postprandial state with CM and do not exchange with other lipoproteins in the plasma. To understand the mechanism of secretion of RE by the intestine under the fasting and postprandial states, differentiated Caco-2 cells were supplemented with radiolabeled retinol under conditions that support or do not support CM secretion. We observed that these cells assimilate vitamin A by a rapid uptake mechanism. After uptake, cells store retinol in both esterified and unesterified forms. Under fasting conditions, cells do not secrete RE but secrete free retinol unassociated with lipoproteins. Under postprandial conditions, cells secrete significant amounts of RE only with CM. The secretion of RE with CM was independent of the rate of uptake of retinol and intracellular free and esterified retinol levels, and was absolutely dependent on the assembly and secretion of CM. The secretion of RE was correlated with the secretion of CM and not with the secretion of total apolipoprotein B. Inhibition of CM secretion by Pluronic L81 decreased the secretion of RE and did not result in their increased secretion with smaller lipoproteins. These data strongly suggest that RE secretion by the intestinal cells is a specific and regulated process that occurs in the postprandial state and is dependent on the assembly and secretion of CM. We propose that RE are added to CM during final stages of lipoprotein assembly and may serve as signposts for these steps.

Effect of brefeldin A on lymphatic triacylglycerol transport in the rat.

Brefeldin A (BFA) has been shown in in vitro studies to either collapse the Golgi into the endoplasmic reticulum (ER) or the peripheral organelles into the trans-Golgi network. Our goal was to determine the effect of BFA on intestinal lipid transport, since the Golgi is thought to play an important role in this process and simultaneously establish the effectiveness of BFA in an in vivo system. We infused rats intraduodenally with glyceryl tri-[3H]oleate at 135 mumol/h for 15 h and included BFA, 750 micrograms/h, during hours 4-7 of infusion. Mass and lipid disintegrations per minute output into the lymph fell to 9% of input rates at 8 h of infusion and returned to steady-state values at 12 h of infusion. Both chylomicron and very low-density lipoprotein output were severely affected by the BFA. Electron microscopy showed that the Golgi was collapsed into the ER. Mucosal triacylglycerol (TG) mass and disintegrations per minute were increased at 7 h of infusion in BFA infused rats vs. controls in the proximal half of the intestine. Lipid absorption, lipase activity, and mucosal TG synthesis were normal in the BFA-treated rats. We conclude that BFA works in vivo and in the intestine collapses the Golgi into the ER. As a consequence, lymphatic TG transport was severely affected.

Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and gp330, two members of the low density lipoprotein receptor gene family, share a multitude of cysteine-rich repeats. LRP has been shown to act as an endocytosis-mediating receptor for several ligands, including protease-antiprotease complexes and plasma lipoproteins. The former include alpha 2-macroglobulin-protease complexes and plasminogen activator inhibitor-activator complexes. The latter include chylomicron remnant-like particles designated beta-very low density lipoproteins (beta-VLDL) complexed with apoprotein E or lipoprotein lipase. The binding specificity of gp330 is unknown. In the current studies we show that gp330 from rat kidney membranes binds several of these ligands on nitrocellulose blots. We also show that both LRP and gp330 bind an additional ligand, bovine lactoferrin, which is known to inhibit the hepatic clearance of chylomicron remnants. Lactoferrin blocked the LRP-dependent stimulation of cholesteryl ester synthesis in cultured human fibroblasts elicited by apoprotein E-beta-VLDL or lipoprotein lipase-beta-VLDL complexes. Cross-competition experiments in fibroblasts showed that the multiple ligands recognize at least three distinct, but partially overlapping sites on the LRP molecule. Binding of all ligands to LRP and gp330 was inhibited by the 39-kDa protein, which co-purifies with the two receptors, suggesting that the 39-kDa protein is a universal regulator of ligand binding to both receptors. The correlation of the inhibitory effects of lactoferrin in vivo and in vitro support the notion that LRP functions as a chylomicron remnant receptor in liver. LRP and gp330 share a multiplicity of binding sites, and both may function as endocytosis-mediating receptors for a large number of ligands in different organs.

Effect of Pluronic L81, a hydrophobic surfactant, on intestinal mucosal cholesterol homeostasis.

Addition of triglyceride and phospholipid to sodium taurocholate when chylomicron output was blocked by L81 did not increase lymphatic total cholesterol output or mucosal unesterified (UC) content more than with sodium taurocholate alone, but mucosal esterified cholesterol (CE) was increased slightly. In these animals with defective chylomicron formation, excess cholesterol accumulated in the intestinal mucosa mainly as CE. The mucosal cholesterol content of animals with normal chylomicron transport expanded during cholesterol and triglyceride absorption, and the expansion led to increased lymphatic secretion of CE. These animals accumulated significantly less CE in their mucosa than did rats treated with L81, but had about the same amount of mucosal UC. However, the overall uptake of cholesterol from the lumen, as determined by either radioactivity or mass of cholesterol in mucosa and lymph, was significantly less in the L81 rats. Also, more radioactive cholesterol remained in the lumen of the L81-treated rats. Finally, the data on specific activities of free and esterified cholesterol showed that the mucosal cholesterol derived from the lumen does not mix evenly with the free cholesterol pool in the enterocytes and is preferentially esterified for export in lymph as triglyceride-rich lipoprotein.