Release Of Chylomicrons Research Articles

Use of Nanovesicles from Orange Juice to Reverse Diet-Induced Gut Modifications in Diet-Induced Obese Mice

We have determined whether orange juice-derived nanovesicles (ONVs) could be used for the treatment of obesity-associated intestinal complications. ONVs were characterized by lipidomic, metabolomic, electron microscopy. In vitro, intestinal barriers (IBs = Caco-2+HT-29-MTX) were treated with ONVs and co-cultured with adipocytes to monitor IB fat release. In vivo, obesity was induced with a high-fat, high-sucrose diet (HFHSD mice) for 12 weeks. Then, half of HFHSD mice were gavaged with ONVs. One-month ONV treatment did not modify HFHSD-induced insulin resistance but reversed diet-induced gut modifications. In the jejunum, ONVs increased villi size, reduced triglyceride content, and modulated mRNA levels of genes involved in immune response (tumor necrosis factor [TNF]-α and interleukin [IL]-1β), barrier permeability (CLDN1, OCLN, ZO1), fat absorption, and chylomicron release. ONVs targeted microsomal triglyceride transfer protein (MTP) and angiopoietin-like protein-4 (ANGPTL4), two therapeutic targets to reduce plasma lipids and inflammation in gastrointestinal diseases. Interestingly, ONV treatment did not aggravate liver steatosis, as MTP mRNA was increased in the liver. Therefore, ONVs protected both intestine and the liver from fat overload associated with the HFHSD. As ONVs concentrated amino acids and bioactive lipids versus orange juice, which are deficient in obese patients, the use of ONVs as a dietary supplement could bring physiological relevant compounds in the jejunum to accelerate the restoration of intestinal functions during weight loss in obese patients.

Emerging Role of Lymphatics in the Regulation of Intestinal Lipid Mobilization.

Intestinal handling of dietary triglycerides has important implications for health and disease. Following digestion in the intestinal lumen, absorption, and re-esterification of fatty acids and monoacylglycerols in intestinal enterocytes, triglycerides are packaged into lipoprotein particles (chylomicrons) for secretion or into cytoplasmic lipid droplets for transient or more prolonged storage. Despite the recognition of prolonged retention of triglycerides in the post-absorptive phase and subsequent release from the intestine in chylomicron particles, the underlying regulatory mechanisms remain poorly understood. Chylomicron secretion involves multiple steps, including intracellular assembly and post-assembly transport through cellular organelles, the lamina propria, and the mesenteric lymphatics before being released into the circulation. Contrary to the long-held view that the intestinal lymphatic vasculature acts mainly as a passive conduit, it is increasingly recognized to play an active and regulatory role in the rate of chylomicron release into the circulation. Here, we review the latest advances in understanding the role of lymphatics in intestinal lipid handling and chylomicron secretion. We highlight emerging evidence that oral glucose and the gut hormone glucagon-like peptide-2 mobilize retained enteral lipid by differing mechanisms to promote the secretion of chylomicrons via glucose possibly by mobilizing cytoplasmic lipid droplets and via glucagon-like peptide-2 possibly by targeting post-enterocyte secretory mechanisms. We discuss other potential regulatory factors that are the focus of ongoing and future research. Regulation of lymphatic pumping and function is emerging as an area of great interest in our understanding of the integrated absorption of dietary fat and chylomicron secretion and potential implications for whole-body metabolic health.

The overall fatty acid absorption controlled by basolateral chylomicron excretion under regulation of p-JNK1

Suppression of fatty acid absorption is one goal to fight obesity. However, the responsible molecular mechanism is poorly understood. Aim of the present study was the search for the key regulator of the overall fatty acid absorption mechanism and its pharmaceutical modulation. As experimental tool we employed the polarized human intestinal tumor derived cell line CaCo2. Here we showed that influx of fatty acids is mediated by an apical heterotetrameric plasma membrane protein complex of which the calcium-independent membrane phospholipase A2 (iPLA2ß) is one constituent. The newly synthesized bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) blocked iPLA2ß, which structurally disrupted the fatty acid-uptake complex. Furthermore, the inhibition of iPLA2ß lead to reduction of cytosolic lysophosphatidylcholine (LPC) production which suppressed p-JNK1, as a central regulator of metabolism. In a concerted action low p-JNK1 levels prohibited synthesis of the members of the fatty acid uptake complex as well as of apolipoprotein B and the connected members of the basolateral vesicular chylomicron excretion machinery, thereby inhibiting cellular lipid excretion. The basolateral chylomicron release was shown to determine the overall fatty acid-absorption capacity as rate limiting step, whereas apical uptake replenishes the cellular stores, enabling continuous transcellular movement of fatty acids. In conclusion, the UDCA-LPE mediated inhibition of p-JNK1 represents a powerful tool to control intestinal absorption of fatty acids and, thus may be employed as a drug to treat obesity.

Glucagon-Like Peptide 2 (GLP-2) Stimulates Postprandial Chylomicron Production and Postabsorptive Release of Intestinal Triglyceride Storage Pools via Induction of Nitric Oxide Signaling in Male Hamsters and Mice.

The intestinal overproduction of apolipoprotein B48 (apoB48)-containing chylomicron particles is a common feature of diabetic dyslipidemia and contributes to cardiovascular risk in insulin resistant states. We previously reported that glucagon-like peptide-2 (GLP-2) is a key endocrine stimulator of enterocyte fat absorption and chylomicron output in the postprandial state. GLP-2's stimulatory effect on chylomicron production in the postabsorptive state has been confirmed in human studies. The mechanism by which GLP-2 regulates chylomicron production is unclear, because its receptor is not expressed on enterocytes. We provide evidence for a key role of nitric oxide (NO) in mediating the stimulatory effects of GLP-2 during the postprandial and postabsorptive periods. Intestinal chylomicron production was assessed in GLP-2-treated hamsters administered the pan-specific NO synthase (NOS) inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), and in GLP-2-treated endothelial NOS knockout mice. L-NAME blocked GLP-2-stimulated apoB48 secretion and reduced triglycerides (TGs) in the TG-rich lipoprotein (TRL) fraction of the plasma in the postprandial state. Endothelial NOS-deficient mice were resistant to GLP-2 stimulation and secreted fewer large apoB48-particles. When TG storage pools were allowed to accumulate, L-NAME mitigated the GLP-2-mediated increase in TRL-TG, suggesting that NO is required for early mobilization and secretion of stored TG and preformed chylomicrons. Importantly, the NO donor S-nitroso-L-glutathione was able to elicit an increase in TRL-TG in vivo and stimulate chylomicron release in vitro in primary enterocytes. We describe a novel role for GLP-2-mediated NO-signaling as a critical regulator of intestinal lipid handling and a potential contributor to postprandial dyslipidemia.

Glucagon-Like Peptide-2 Regulates Release of Chylomicrons From the Intestine

The intestine efficiently incorporates and rapidly secretes dietary fat as chylomicrons (lipoprotein particles comprising triglycerides, phospholipids, cholesterol, and proteins) that contain the apolipoprotein isoform apoB-48. The gut can store lipids for many hours after their ingestion, and release them in chylomicrons in response to oral glucose, sham feeding, or unidentified stimuli. The gut hormone glucagon-like peptide-2 (GLP-2) facilitates intestinal absorption of lipids, but its role in chylomicron secretion in human beings is unknown. We performed a randomized, single-blind, cross-over study, with 2 study visits 4 weeks apart, to assess the effects of GLP-2 administration on triglyceride-rich lipoprotein (TRL) apoB-48 in 6 healthy men compared with placebo. Subjects underwent constant intraduodenal feeding, with a pancreatic clamp and primed constant infusion of deuterated leucine. In a separate randomized, single-blind, cross-over validation study, 6 additional healthy men ingested a high-fat meal containing retinyl palmitate and were given either GLP-2 or placebo 7 hours later with measurement of TRL triglyceride, TRL retinyl palmitate, and TRL apoB-48 levels. GLP-2 administration resulted in a rapid (within 30 minutes) and transient increase in the concentration of TRL apoB-48, compared with placebo (P = .03). Mathematic modeling of stable isotope enrichment and the mass of the TRL apoB-48 suggested that the increase resulted from the release of stored, presynthesized apoB-48 from the gut. In the validation study, administration of GLP-2 at 7 hours after the meal, in the absence of additional food intake, robustly increased levels of TRL triglycerides (P = .007), TRL retinyl palmitate (P = .002), and TRL apoB-48 (P = .04) compared with placebo. Administration of GLP-2 to men causes the release of chylomicrons that comprise previously synthesized and stored apoB-48 and lipids. This transiently increases TRL apoB-48 levels compared with placebo. Clinical trials number at www.clinicaltrials.gov: NCT 01958775.

The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium

The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine (Arfrp1vil−/−) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. Arfrp1vil−/− enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the Arfrp1vil−/− epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylo­microns and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells.

Mechanisms Involved in the Intestinal Digestion and Absorption of Dietary Vitamin A

Dear Editor: Harrison and Hussain (1) elegantly summarized the current knowledge concerning the metabolism of dietary vitamin A during digestion and absorption on the enteral level. They showed that absorption and enteral processing and chylomicron release into the body is highly regulated. They concluded that no lipoproteins other than chylomicrons and lipoproteins of the VLDL fraction can transport retinyl ester in plasma. This indeed is peculiar because other lipids and lipophilic components, such as triglycerides, cholesterol, phospholipids, vitamin E and carotenoids, are found in all lipoprotein fractions. We and others have shown that within the animal kingdom, retinyl esters occur physiologically at high concentrations in blood plasma not only in dogs but also in many other carnivorous species (2–4). In ferrets and dogs, the majority of retinyl esters (primarily retinyl stearate and palmitate) are associated with the HDL fraction (5,6). Although their plasma levels are dependent on the oral supplementation of vitamin A, substantial levels are still present when animals were fed a vitamin A-depleted diet for an extended period of time (7,8). The importance of retinyl esters in the pathogenesis of vitamin A intoxication is controversial (9,10). In humans with a two-point mutation in the DNA sequence of the retinol binding protein gene, no effects other than night blindness occur despite extremely low levels of retinol. This observation suggests that the usual cellular supply of retinol might be bypassed by retinyl esters and/or -carotene or retinoic acid (11). Results in carnivorous species might further support the hypothesis that retinyl esters are of additional importance for the supply of peripheral target tissues. It remains to be answered why only carnivores’ plasma retinyl esters behave similarly to triglycerides and cholesteryl esters but not in any other species in which mainly retinol is present in plasma. The molecular and cellular bases of the lipoprotein associated transport of retinyl esters in plasma as well as the excretion of substantial amounts of retinol and retinyl esters in the urine of carnivores (3) remain to be elucidated. In both cases, however, it seems obvious that plasma transport and urinary excretion are not only dependent on dietary supplementation but are highly regulated (7). This regulation might involve both the liver and the kidney. Carnivores might, thus, provide an interesting model to study the importance of the gut and the liver in the regulation of the incorporation of retinyl esters into lipoproteins other than chylomicrons and the kidney in the uptake and the regulated excretion of retinyl esters in the urine.

Rat intestinal alpha1-antitrypsin secretion is regulated by triacylglycerol feeding.

alpha1-Antitrypsin (AAT) is secreted by the enterocyte, but its regulation of expression, intramucosal distribution, and functional status are unclear. After corn oil gavage (plus Pluronic L-81 to block chylomicron release), rat intestine was examined for mRNA encoding AAT, immunoreactivity by light and electron microscopy, and protein content by Western blot. Species-specific antisera used were raised against both AAT and surfactant-like particle (SLP), a membrane secreted by the enterocyte in response to fat feeding. Purified luminal SLP was fractionated by Bio-Gel P-200 chromatography to assess its interaction with AAT. Triacylglycerol feeding maximally increased mucosal mRNA-encoding AAT and AAT intracellular protein content by 3 and 5 h, respectively. Immunocytochemistry revealed predominance of AAT in basolateral spaces around enterocytes and Pluronic-blocked extracellular accumulation of AAT, patterns nearly identical to those of secreted SLP. About 10% of AAT was reversibly associated with SLP. Luminal AAT was smaller (51 kDa) than mature AAT (55 kDa) and did not form a complex with pancreatic elastase. When the common bile duct was tied, excluding pancreatic proteases from the lumen, mature AAT that was cleaved by pancreatic elastase was secreted. The luminal secretion of AAT and its reversible association with SLP suggest an intracellular association and a possible role for AAT during lipid digestion and absorption.

Role of lymph flow in intestinal chylomicron transport.

In this study we investigated the influence of lymph flow on chylomicron transport. We examined the effects of varying the hydration of the interstitial matrix on chylomicron appearance time and on lymphatic lipid transport rate when a lipid test meal containing oleic acid and 1-monoolein was infused intraduodenally at a constant rate. The three groups of rats tested were control rats (normal interstitial hydration), rats receiving intravenous saline infusion (expanded interstitial matrix), and rats with an attenuated water absorption rate (dehydrated interstitial matrix). This study shows that lymph flow has a profound effect on intestinal chylomicron transport. As lymph flow increased, the chylomicron appearance time (time between the placement of radioactive fatty acid into the intestinal lumen to the appearance of radioactive lipid in the central lacteal) was reduced. When lymph flow exceeded 40 microliter/min, the chylomicron appearance time reached a minimum value of 13.6 min. This minimum chylomicron appearance time probably represents the time required for assembly of absorbed lipid, formation of chylomicrons, and their subsequent discharge into the lymphatics. The chylomicron appearance time lengthened as lymph flow fell. The results of this study underscore the necessity of using steady-state lymphatic lipid output data to assess factors affecting the cellular packaging and release of chylomicrons in the small intestine.

Cytochemistry of Fat Absorption

Publisher Summary After ingestion of a fat-containing meal, dietary lipids are partially hydrolyzed and absorbed into intestinal mucosa. In the mucosa, the hydrolytic products are resynthesized into triglycerides, cholesterol, and phospholipids. The resulting lipids are combined with apoproteins to form intestinal lipoproteins, primarily chylomicrons, and very low-density lipoproteins. The chapter discusses the mechanisms of the absorption of triglyceride—the major source of lipids in the diet. The mechanism of chylomicron secretion from the intestinal absorptive cells into the intercellular space has eluded precise ultrastructural definition. The secretion of nascent chylomicrons is thought to involve exocytosis or the fusion of lipid-laden secretory vesicles with the lateral plasmalemma, resulting in the release of chylomicrons into the intercellular spaces. The chapter provides a general morphology of the rat intestinal epithelial cell. The intestinal epithelial cell is tall and columnar. The apical border is largely covered by an array of regularly spaced microvilli, which may increase the mucosal surface. The basal surface of the cell rests on a thin basement membrane that separates the epithelium from the lamina propria.

Size of Lipoprotein Particles in the Intestinal Lymph of Rats Fed on Corn Oil, Peanut Oil, Rapeseed Oil or Canbra Oil

The release of chylomicrons (d > 0.1 µm) and very low density lipoprotein-like particles (d ≤ 0.1 µm) by the intestinal mucosa of rats was followed by means of electron microscopy of the intestinal lymph after ingestion of four oils: corn and peanut oils, both containing large amounts of C18 unsaturated fatty acids (85 and 82%, respectively), especially linoleic acid (50%) in the corn oil and oleic acid (53%) in the peanut oil, and two crucifer oils: rapeseed oil, with a high erucic content (53%) and canbra oil with alow erucic content but rich in oleic acid (62%). The absorption of rapeseed oil took place at a very early stage and was very rapid; practically within the first 6 h. The absorption of corn oil also began early but it took longer. As for canbra and peanut oils, their absorption occurred later and evolved in the same manner but with, however, greater rapidity for the canbra oil. The proportion of chylomicrons observed in lymph following the ingestion of canbra oil, especially those larger than 0.2 µm, was higher than the proportion observed after ingestion of peanut oil, but the release of chylomicrons during the course of absorption of either oil followed the same evolution. Most of the fatty acids of these two oils were thus transported by chylomicrons, especially after ingestion of canbra oil. A delayed release of chylomicrons occurred after the ingestion of rapeseed oil. Since the absorption of this oil began very early and increased rapidly, most of the fatty acids, particularly erucic acid, were carried in small chylomicrons and by very low density lipoprotein (VLDL)-like particles. The large proportion and the rapidity of the release of chylomicrons after ingestion of peanut oil and particularly canbra oil could be ascribed to the oleic acid content of these two oils. The results concerning the absorption of rapeseed oil could be explained by the saturated character of erucic acid. The incorporation of large amounts of this acid in the smaller lipoprotein particles of lymph could partly explain the abnormal metabolism of erucic acid in the tissues, especially in the myocardium, different from that observed for oleic acid in canbra oil, which is mainly transported in chylomicrons.

Ultrastructural features of regional differences in chylomicron secretion by rat intestine

An electron microscopic study of the effects of prolonged intraduodenal triolein perfusion on intestinal fat absorption was performed to investigate the ultrastructural features of regional differences in chylomicron secretory capacity in the rat small intestine. Utilizing conditions in which intraluminal triolein concentrations were high enough to saturate the mechanism for fat uptake it was demonstrated that the proximal segments reach a steady state of uptake and chylomicron secretion within one hour whereas triglycerides accumulate progressively in the distal epithelial cells. After one or four hours perfusion with [1- 14C]triolein the mucosal triglyceride content was much greater in distal than in proximal segments and electron microscopy disclosed many more and larger intracellular fat droplets in the distal absorptive cells. Six hours after stopping the 4 hr perfusion many distal cells were still filled with large fat droplets whereas the proximal cells were devoid of fat. In comparison to proximal intestine, triglycerides accumulated in distal segments following direct segmental triolein perfusion and electron microscopy disclosed large intracellular fat droplets similar to those observed after intraduodenal triolein perfusion. The ultrastructural observations suggest that fat accumulation, in the distal segments, is due to a relatively decreased capacity for chylomicron release. These studies suggest an intrinsic difference between proximal and distal intestine in fat absorption, which may reflect regional differences in cellular function such as membrane availability for the final assembly and secretion of chylomicrons.

Lepid transport across the intestinal epithelial cell: Effect of colchicine

Rats injected with colchicine (0.5 mg/100 g of body weight) 1 h before ingestion of a margarine emulsion (1 g in 2 ml of saline) do not show the rise in plasma triacylglycerol concentration found in controls during the subsequent hours. The effect of colchicine is more dramatic when the experiment is performed after prior administration of Triton WR-1339, a substance known to inhibit the catabolism of lipoproteins. Colchicine-treated rats also showed a fivefold increase in the content of triacylglycerol in proximal jejunum, when compared to controls. These results are consistent with the idea that colchicine interferes with the intracellular phase of fat absorption, suggesting that the microtubular-microfilamentous system could be involved in the release of chylomicrons from the intestinal cell into the circulation.

Protein and glycerolipid biosynthesis in isolated intestinal epithelial cells of normal and bile-fistula rats

Protein and glycerolipid biosynthesis was studied in isolated epithelial cells of the intestinal mucosa of normal and 1-day bile-fistula rats. In cells from fistula rats, protein synthesis from [1- 14C]leucine was decreased 40–45% and glycoprotein synthesis from [1- 14C]glucosamine 25–30%. Under comparable conditions, the synthesis of phosphatidylcholine from a variety of precursors was decreased 40–45% while no change was observed in the formation of either phosphatidylethanolamine or triacylglycerols. Chylomicron release was inhibited 70–80% in the cells from the bile-fistula rats. In vitro addition of either lysophosphatidylcholine or choline to the cells from the bile-fistula animals resulted in greatly increased phosphatidylcholine and protein biosynthesis and an effective release of chylomicrons. It is suggested that this stimulation of incorporation of label is due to a net synthesis of phosphatidylcholine required for membrane and lipoprotein repairs in the fistula cells. These results provide further evidence, that, in the rat, biliary phosphatidylcholine may play an essential role in the formation and clearance of chylomicrons from the intestinal mucosa.

Effect of puromycin in vitro on protein and glycerolipid biosynthesis in isolated epithelial cells of rat intestine

1. 1. The in vitro effect of puromycin on protein and glycerolipid biosynthesis was studied in isolated epithelial cells of the small intestine of the rat. 2. 2. Puromycin caused a 85–90% inhibition of protein synthesis but only a 10–20% suppression of glycoprotein formation after one hour. 3. 3. Triacylglycerol synthesis from glycerol and 2-hexadecylglycerol was unaffected while a 30–35 % inhibition of phosphatidylcholine formation from glycerol and lysophosphatidylcholine occurred after one hour. In the same time, chylomicron release was also inhibited by 30–35 %. 4. 4. Except for protein biosynthesis, which was similarly affected, the inhibitory effects of puromycin were markedly smaller in vitro than those seen previously after puromycin administration in, vivo. 5. 5. It is suggested that the inhibition of phospholipid biosynthesis by puromycin results largely from its depressing effect upon the biogenesis of the endoplasmic reticulum.

Release of chylomicrons by isolated cells of rat intestinal mucosa.

AbstractFat‐laden mucosal cells were isolated by flotation from fed male rats after digesting scrapings of washed jejunum with collagenase in bicarbonate buffer. About 50–60 million cells were obtained per preparation, which were 95–100% viable as assessed by Trypan Blue. The isolated cells were capable of effective incorporation of labeled fatty acids and glucose into triglycerides and phospholipids, and of labeled leucine and glucosamine into the protein envelope of the released chylomicrons. The secretion of the labeled protein paralleled the release of the labeled fat, both of which were linear with the concentration of the albumin in the incubation mixture. About 80% of the total fat of the cell was released as chylomicrons within 30 min when incubated in the presence of albumin‐bicarbonate buffer. Injection of puromycin 24 hr prior to harvesting of cells led to a complete inhibition of chylomicron release. Addition of puromycin to the incubation medium gave 50–80% inhibition of release. No inhibition of release of chylomicrons resulted from a treatment with ethionine. The released chylomicrons were separated from the cells by Millipore filtration.

Does puromycin block release of chylomicrons from the intestine?

Does puromycin block release of chylomicrons from the intestine?