Ultrastructural demonstration of the absorption and transportation of minute chylomicrons by subepithelial blood capillaries in rat jejunal villi.

Chylomicrons from villous columnar epithelial cells are generally known to be transported only by central lymph vessels (CLV), whereas antigenic particulates derived from the intestinal lumen can also be transported by subepithelial blood capillaries (sBCs) in rat intestinal villi. The possibility of chylomicron absorption by sBCs was histoplanimetrically studied in the rat jejunum under a transmission electron microscope. The chylomicrons more abundantly presented in villous venules than in arterioles. The most frequent size (MFS) of chylomicrons was 75 to 90 nm in diameter in the areas near sBCs, while it was 45 to 60 nm in the epithelial intercellular spaces just above sBCs or the intermediate areas between sBCs. The MFS of chylomicrons was 45 to 60 nm in the intermediate areas between sBCs and in the epithelial intercellular spaces just above these areas. The MFS of chylomicrons in CLV was intermediate between that in the area adjacent to sBCs and that in the intermediate areas between sBCs. Chylomicrons were found in small vesicles in the endothelial cytoplasms of sBCs. No chylomicrons larger than 600 nm were observed in the lamina propria. These findings suggest that some of the chylomicrons smaller than 75 nm, which are probable intestinal very low-density lipoproteins (VLDL), are directly transported to the liver by hepatic portal blood in addition to CLV and that epithelial fat droplets larger than 600 nm are not discharged into lamina propria in rat jejunum under physiological conditions.

Uptake of postprandial lipoproteins into bone in vivo: impact on osteoblast function.

Dietary lipids and lipophilic vitamins are transported by postprandial lipoproteins and are required for bone metabolism. Despite that, it remains unknown whether bone cells are involved in the uptake of circulating postprandial lipoproteins in vivo. The current study was performed to investigate a putative participation of bone in the systemic postprandial lipoprotein metabolism in mice, to identify potentially involved cell type populations and to analyze whether lipoprotein uptake affects bone function in vivo. As a model for the postprandial state, chylomicron remnants (CR) were injected intravenously into mice. Next to the liver and compared to other organs, bone appeared to be the second most important organ for the clearance of radiolabeled CR particles from the circulation in vivo. In addition, uptake of radiolabeled CR by primary murine osteoblasts and hepatocytes was quantified to be in a similar range in vitro. A complementary approach with fluorescently labeled CR and immunohistochemical staining for apoE proved that intact CR particles were taken up into bone and liver. Electron microscopy localization studies of bone sections revealed CR uptake into sinusoidal endothelial cells, macrophages and osteoblasts. The relative amount of radiolabeled CR uptake into femoral cortical bone, representing predominantly osteoblasts, and bone marrow, representing predominantly non-osteoblast cells, was within the same range. Most importantly, the injection of vitamin K1-enriched CR resulted in an increase of the degree of osteocalcin carboxylation in vivo while total osteocalcin concentrations remained unaffected, giving functional proof that osteoblasts process CR in vivo. In conclusion, here we demonstrate that bone is involved in the postprandial lipoprotein metabolism in mice. Osteoblasts participate in CR clearance from the circulation, which has a direct impact on the secretory function of osteoblasts.

Elucidation of lipoprotein particles structure by proteomic analysis.

Lipoproteins are responsible for lipid packaging and transport through the bloodstream, and for their delivery to target tissues. Their participation in process, such as inflammation and innate immunity has also been suggested recently. Lipoprotein particles have very complex biochemical structures, which result from intricate processes involving coordinated mechanisms of protein and lipid synthesis, intracellular assembling and trafficking, and intra- and extracellular metabolism. Alterations in these mechanisms cause several negative effects on human health. The ability of current proteomic approaches to dissect the dynamic nature of complex particles revealing protein composition and post-translational modifications is shedding further light on lipoprotein structures and functions. This review summarizes lipoprotein classification, biogenesis and metabolism as well as discussing how the results of 20 proteomics-based reports integrate our knowledge on both their biochemical composition and their effects on target cells, thus contributing to reveal the possible functions.

A histological, histochemical and ultrastructural study of the digestive tract of Dentex dentex (Pisces, Sparidae).

Dentex dentex has a short esophagus, a large caecal type stomach, three to six pyloric caeca and a short intestine. Light and electron microscope studies reveal that the esophageal mucosa displays primary and secondary folds, a stratified squamous epithelium with fingerprint-like microridges alternating with a few zones formed by a single layer of columnar cells with apical microvilli. Only primary folds are present in the stomach, which is rich in simple tubular glands, these being absent in the pyloric valve. Two cell types occur in the gastric glands, one with a well developed apical intracytoplasmic membrane system consisting of a vesicular network of smooth membranes, and the other with a supranuclear tubulovesicular system. Pyloric caeca and anterior and posterior intestine mucosae display the same pattern of folding, with primary and secondary folds, without following a definite pattern in their orientation. In the rectum, the folds are oriented longitudinally. Small dense particles containing chylomicrons appear in groups in the intercellular spaces of the caecal and anterior intestinal epithelia. Eosinophilic granular cells (mast cells) appear along the digestive tract mainly within the stratum compactum. Histochemical studies reveal no differences in the composition of goblet cell mucus along the digestive tract. No histochemical differences were detected between enterocytes of the intestine, pyloric caeca and rectum. Neutral mucosubstances dominate in the stomach epithelium and in the goblet cells of the esophagus, pyloric caeca and anterior intestine. Results of the present study are discussed in relation to descriptions of the digestive tract in other sparids.

Lymphatic chylomicron size is inversely related to biliary phospholipid secretion in mice.

Biliary phospholipids (PL) stimulate dietary fat absorption by facilitating intraluminal lipid solubilization and by providing surface components for chylomicron (CM) assembly. Impaired hepatic PL availability induces secretion of large very-low-density lipoproteins, but it is unclear whether CM size depends on biliary PL availability. Biliary PL secretion is absent in multidrug resistance protein 2-deficient (Mdr2(-/-)) mice, whereas it is strongly increased in essential fatty acid (EFA)-deficient mice. We investigated lymphatic CM size and composition in mice with absent (Mdr2(-/-)) or enhanced (EFA deficient) biliary PL secretion and in their respective controls under basal conditions and during enteral lipid administration. EFA deficiency was induced by feeding mice a high-fat, EFA-deficient diet for 8 wk. Lymph was collected by mesenteric lymph duct cannulation with or without intraduodenal lipid administration. Lymph was collected in 30-min fractions for up to 4 h, and lymphatic lipoprotein size was determined by dynamic light-scattering techniques. Lymph lipoprotein subfractions were isolated by ultracentrifugation, and lipid composition was measured. Lymphatic CMs were significantly larger in Mdr2(-/-) mice than in Mdr2(+/+) controls either without (+50%) or with (+25%) enteral lipid administration, and molar core-surface ratios were increased [triglyceride (TG)-to-PL ratio: 4.4 +/- 1.4 in Mdr2(-/-) mice vs. 2.7 +/- 0.8 in Mdr2(+/+) mice, P < 0.001]. In contrast, EFA-deficient mice secreted lipoproteins into lymph that were significantly smaller than in EFA-sufficient controls (173 +/- 32 vs. 236 +/- 47 nm), with correspondingly decreased core-surface ratios (TG-to-PL ratio: 3.0 +/- 1.0 in EFA-deficient mice vs. 6.0 +/- 1.9 in EFA-sufficient mice, P < 0.001). CM size increased during fat absorption in both EFA-deficient and EFA-sufficient mice, but the difference between the groups persisted. In conclusion, the present results strongly suggest that the availability of biliary PL is a major determinant of the size of intestinally produced lipoproteins both under basal conditions and during lipid absorption. Altered CM size may have physiological consequences for postprandial CM processing.

Effect of a surfactant, Tween 80, on the formation and secretion of chylomicrons in the rat.

Effects of Tween 80, a nonionic surfactant, on size and number of chylomicrons (CM) secreted during lipid absorption in the rat model are reported. Fasted rats were loaded with either 154 mM NaCl or 25% (w/w) olive oil emulsion in 154 mM NaCl with 0, 0.1, 1 or 10% (w/w) Tween 80. After 3 h, either mesenteric lymph or blood was collected and their triacylglycerol-rich lipoprotein fraction (Sf>20 and Sf>400, respectively) isolated. Triacylglycerol (TAG) and apolipoprotein B48 (apoB48) concentrations in the lipoprotein fractions were assayed and their fluxes (lymph) calculated. TAG:apoB48 ratios, indicative of CM size, were determined. The data support the hypothesis that fat loading is accommodated mainly by increased average size, rather than number, of CM. Co-administration of Tween 80 with olive oil resulted in a significant increase in CM apoB48 secretion into the mesenteric lymph duct and in an increased concentration of apoB48 in the blood (Sf>400). Also, Tween 80-treated groups exhibited smaller mean CM size relative to the olive oil only group in both lymph and blood. The observed effect on CM size and number did not appear to be dose dependent at concentrations of Tween 80 above 0.1% (w/w). Incorporation of Tween 80 in the diet at 1 or 10% (w/w) concentrations reduced the TAG concentration in the stools; however, a significant increase in water content was observed at 10% (w/w) concentration. In conclusion, Tween 80 at 1 or 10% (w/w) doses can improve the efficiency of the digestive system to absorb dietary fat but at high concentrations (10%, w/w) it appeared to have a toxic or irritating effect on the gastrointestinal system. More importantly, the effect of Tween 80 on size and number of CM is a condition that favours a delay in their clearance rate.

COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.

The budding of vesicles from endoplasmic reticulum (ER) that contains nascent proteins is regulated by COPII proteins. The mechanisms that regulate lipid-carrying pre-chylomicron transport vesicles (PCTVs) budding from the ER are unknown. To study the dependence of PCTV-ER budding on COPII proteins we examined protein and PCTV budding by using ER prepared from rat small intestinal mucosal cells prelabeled with (3)H-oleate or (14)C-oleate and (3)H-leucine. Budded (3)H-oleate-containing PCTVs were separated by sucrose density centrifugation and were revealed by electron microscopy as 142-500 nm vesicles. Our results showed the following: (1) Proteinase K treatment did not degrade the PCTV cargo protein, apolipoprotein B-48, unless Triton X-100 was added. (2) PCTV budding was dependent on cytosol and ATP. (3) The COPII proteins Sar1, Sec24 and Sec13/31 and the membrane proteins syntaxin 5 and rBet1 were associated with PCTVs. (4) Isolated PCTVs were able to fuse with intestinal Golgi. (5) Antibodies to Sar1 completely inhibited protein vesicle budding but increased the generation of PCTV; these changes were reversed by the addition of recombinant Sar1. (6) PCTVs formed in the absence of Sar1 did not contain the COPII proteins Sar1, Sec24 or Sec31 and did not fuse with the Golgi complex. Together, these findings suggest that COPII proteins may not be required for the exit of membrane-bound chylomicrons from the ER but that they or other proteins may be necessary for PCTV fusion with the Golgi.

Chylomicron retention disease--the role of ultrastructural examination in differential diagnosis.

Three children with malabsorption presumably caused by celiac disease had undergone jejunal biopsy. While a histological examination revealed microvacuolization of enterocytes in the absence of celiac lesions, an ultrastructural investigation disclosed numerous chylomicrons and larger lipid vacuoles inside the cytoplasm of enterocytes, mostly in the supranuclear region. No chylomicrons were evident in the interstitium between adjacent enterocytes, as observed in normal subjects. These ultrastructural findings allowed for the diagnosis of "Chylomicron retention disease" (CRD). CRD was described for the first time by Anderson in 1961, and it is included in the group of disorders of biosynthesis and secretion of B apolipoproteins (apoB). This disease, in particular, appears to result from a specific defect involving the secretion of lipoproteins containing apoB-48 from the gut, with the complete absence of post prandial chylomicrons in the sera. CRD needs to be recognized early because of its adverse effects on growth and its potential for neurological and ocular complications, and the ultrastructural identification of chylomicron-size lipid droplets clustered in the enterocytes, with the absence of fat outside the cells, represents the gold standard to identify CRD. together with clinical aspects and laboratory measurements. In this study, we describe the histological and ultrastructural aspects observed in three pediatric cases of CRD.

Vitamin E deficiency due to chylomicron retention disease in Marinesco-Sjögren syndrome.

We report on 2 brothers (aged 19 and 12 years) with Marinesco-Sjögren syndrome who also had very low serum vitamin E concentrations with an absence of postprandial chylomicrons. The molecular study ruled out ataxia with isolated vitamin E deficiency, abetalipoproteinemia, and hypobetalipoproteinemia. The electron microscopy of the intestinal mucosa was consistent with a chylomicron retention disease. We speculate that both chylomicron retention disease and Marinesco-Sjögren syndrome are related to defects in a gene crucial for the assembly or secretion of the chylomicron particles, leading to very low serum levels of vitamin E.

Chylomicron-sized lipid particles are formed in the setting of apolipoprotein B deficiency.

The mechanisms for packaging large quantities of neutral lipids into apolipoprotein (apo) B-containing lipoproteins (chylomicrons or VLDL) are incompletely understood. However, several lines of evidence have suggested that the addition of core lipids to apoB to form a lipoprotein particle within the endoplasmic reticulum (ER) may involve two steps: first, the addition of small amounts of core lipids to membrane-bound apoB, generating a lipid-poor, small apoB-containing particle, and second, the fusion of that particle with a larger, independently formed triglyceride-rich and apoB-free "lipid particle." We sought to test this two-step hypothesis of apoB core lipidation by using electron microscopy to compare chylomicron assembly in mice that are genetically deficient in the ability to synthesize apoB in the intestine to control mice. In 19-day gestational mice (fasting setting) that were deficient in intestinal apoB synthesis, chylomicron-sized lipid particles in the lumen of the enterocyte ER were even more abundant and were 2- to 3-fold larger than those in the enterocytes of normal control mice. However, there were fewer lipid-staining particles in the Golgi apparatus, and many fewer particles in the extracellular space, compared with normal control mice. In both types of newborn suckling mice, much larger lipid particles were assembled within the lumen of the ER. They were however, less abundant and rarely reached the Golgi apparatus in fatty enterocytes of intestines deficient in apoB synthesis. These observations provide in vivo evidence that chylomicron formation could involve the synthesis of apoB-free triglyceride-rich particles within the endoplasmic reticulum (ER) lumen, and that the transport of these lipid particles out of the ER to Golgi apparatus and interstitium is facilitated by the acquisition of apoB.

A single copy of apolipoprotein B-48 is present on the human chylomicron remnant.

Individuals homozygous for the e2 allele encoding apolipoprotein E exhibit a remnant removal defect and accumulate substantial levels of intestinally derived particles containing apolipoprotein B-48 (apoB-48). Such lipoproteins were isolated from the plasma of E2/E2 individuals, and further purified by affinity chromatography using a polyclonal antibody specific for selective binding and removal of apoB-100-containing lipoproteins. The unbound lipoproteins, termed chylomicron remnants, were particles with average hydrated diameters of 31.2 nm as determined by dynamic light scattering. They contained apoB-48 and ApoE as their only protein components. The number of apoB-48 molecules on each lipoprotein was assessed by counting the number of antibody molecules bound to the surface of the chylomicron remnants, using either a monoclonal antibody specific for a single epitope on apoB-48 or a mixture of two such monoclonal antibodies specific for widely separated epitopes. The results of this analysis seem unambiguous: no more than one apoB-48 resides on the chylomicron remnant. Because apoB appears to be unable to transfer among lipoprotein particles, it may be inferred that nascent chylomicrons also contain a single copy of apoB-48.

Metabolic fate of chylomicrons obtained from rats maintained on diets varying in fatty acid composition.

The importance of the fatty acid component in the metabolism of chylomicrons was demonstrated by feeding diets varying in fatty acid composition which resulted in chylomicrons of different sizes. On a diet rich in polyunsaturated fatty acids (PUFA) from safflower oil, chylomicrons of diameter 1853 +/- 192 A were harvested from the mesenteric lymph, whereas on coconut oil and medium-chain triglyceride diets the chylomicron size was 1403 +/- 83 and 604 +/- 40 A, respectively. When the isolated chylomicrons were injected into recipient rats maintained on a regular diet, their half-life (t1/2) decreased from 5.4 +/- 0.4 to 1.8 +/- 0.3 min with the increase in particle size. No significant difference in the apolipoprotein profile of chylomicrons of various sizes was noted, indicating that alterations of chylomicron removal are not related to apolipoprotein composition. Rats maintained on PUFA diets showed a marked increase in their adipose tissue lipoprotein lipase activity. The fast removal of large chylomicrons and increased tissue lipoprotein lipase activity, together with suppression of hepatic lipogenesis on this diet, apparently explains the low plasma triglyceride level in rats maintained on diets rich in PUFAs.

Size and shape determination of fixed chylomicrons and emulsions with fluid or solid surfaces by three-dimensional analysis of shadows.

Chylomicrons and chylomicron-sized emulsions are spherical particles in suspension but their shape and apparent size may be distorted by electron microscopy processing. To assess adsorption to grids, flattening, and shrinkage, chylomicrons and emulsions were fixed with osmium tetroxide and together with polystyrene beads were shadowed with platinum. Vertical profiles projected from particle shadows indicated that the chylomicrons and emulsions were slightly shrunken, slightly truncated, oblate spheroids while the polystyrene beads were spheres. Particle diameters were corrected by assuming that volumes of oblate spheroids on the grid surface were equal to volumes of spheres in the original lipid suspension. Because of the compensating effects of shrinkage (decreases diameter) and flattening (increases diameter) the differences between the means of measured diameters and corrected diameters were less than or equal to 5%.

Chylomicron synthesis in experimental nephrotic syndrome.

Mesenteric lymph was collected for 48 h from rats with aminonucleoside-induced nephrotic syndrome, receiving an intraduodenal infusion of a triacylglycerol emulsion. In nephrosis, the rates of lymph flow and triacylglycerol transport were approx. 2-fold higher, but the transport of total protein and of apoproteins A-I and E was 2- to 3-fold lower than that in control rats, resulting in chylomicrons with a 3-fold approx. elevated triacylglycerol/protein ratio. Supplementation of the triacylglycerol infusate with glucose and amino acids did not increase the protein or apoA-I and apoE transport. Production or transport of B and C apoproteins in nephrotic rats was also reduced, as indicated by tetramethylurea solubility, incorporation of intraduodenally infused [3H]leucine and staining of the chylomicron proteins on SDS-PAGE gels. Apoprotein A-IV was the only chylomicron component into which the leucine incorporation was elevated, but its relative content was not increased on SDS-PAGE gels. Lymph chylomicrons of nephrotic rats were larger in size (1498 +/- 37 vs. 1235 +/- 23 A), consistent with the higher triacylglycerol/protein ratio. The concentration of all lipoprotein classes was markedly elevated in the plasma of nephrotic rats, as was that of the total A-I and E apoproteins. Intravenous injection of 125I-labelled HDL, followed by tracing of the label in lymph chylomicrons, indicated a lower rate of transfer of HDL apoproteins from plasma to lymph in nephrotic rats. We conclude that the intestinal chylomicron formation in nephrosis is characterised by an enhanced triacylglycerol transport without the appropriate apoprotein complement. This is probably due to the limited capacity of enterocytes, in marked contrast to hepatocytes, to respond to the hypoproteinemia of nephrosis with increased production and/or transport of the apoproteins.

Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits. Factors that modify tissue-specific uptake.

The metabolism of [14C]cholesterol- and [3H]retinol-labeled chylomicrons obtained from canine thoracic duct or rabbit mesenteric lymph was investigated in normal fasted rabbits. Typically, 70-80% of the chylomicrons injected into the rabbits were cleared from the plasma in 20 min, and their uptake was accounted for principally by the liver and the bone marrow. Surprisingly, the bone marrow was a major site of uptake; the uptake ranged from about half that of the liver to a nearly equal amount. The importance and specificity of chylomicron-chylomicron remnant uptake by the bone marrow were established by demonstrating that (a) bone marrow throughout the body accumulated these lipoproteins, (b) the level of uptake was consistent regardless of how the values were calculated or how the chylomicrons were prepared, (c) the uptake represented specific binding, and (d) radiolabeled intestinal lipoproteins induced in vivo delivered cholesterol and retinol to the marrow. Electron microscopic examination of the rabbit bone marrow established that perisinusoidal macrophages uniquely accounted for the uptake of the chylomicrons. Whereas liver cleared a variety of both triglyceride-rich lipoproteins (chylomicrons, chylomicron remnants, and very low density lipoproteins) and cholesterol-rich lipoproteins (beta-very low density lipoproteins and high density lipoproteins containing apolipoprotein E), bone marrow uptake appeared to be restricted to the triglyceride-rich lipoproteins. More chylomicron remnants (generated in a hepatectomized rabbit) were cleared by the liver than by the bone marrow, and the addition of excess apolipoprotein E to chylomicrons resulted in their preferential uptake by the liver. The role of chylomicron-chylomicron remnant delivery of lipids or lipid-soluble vitamins to rabbit bone marrow is open to speculation, and whether triglyceride-rich lipoprotein uptake occurs to a significant extent in the bone marrow of humans remains to be determined.