Intestinal Lipoprotein Synthesis Research Articles

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion.

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating naïve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

Using primary murine intestinal enteroids to study dietary TAG absorption, lipoprotein synthesis, and the role of apoC-III in the intestine

Since its initial report in 2009, the intestinal enteroid culture system has been a powerful tool used to study stem cell biology and development in the gastrointestinal tract. However, a major question is whether enteroids retain intestinal function and physiology. There have been significant contributions describing ion transport physiology of human intestinal organoid cultures, as well as physiology of gastric organoids, but critical studies on dietary fat absorption and chylomicron synthesis in primary intestinal enteroids have not been undertaken. Here we report that primary murine enteroid cultures recapitulate in vivo intestinal lipoprotein synthesis and secretion, and reflect key aspects of the physiology of intact intestine in regard to dietary fat absorption. We also show that enteroids can be used to elucidate intestinal mechanisms behind CVD risk factors, including tissue-specific apolipoprotein functions. Using enteroids, we show that intestinal apoC-III overexpression results in the secretion of smaller, less dense chylomicron particles along with reduced triacylglycerol secretion from the intestine. This model significantly expands our ability to test how specific genes or genetic polymorphisms function in dietary fat absorption and the precise intestinal mechanisms that are critical in the etiology of metabolic disease.

Exon Skipping of Hepatic APOB Pre-mRNA With Splice-switching Oligonucleotides Reduces LDL Cholesterol In Vivo

Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.

Physiological and molecular responses to dietary phospholipids vary between fry and early juvenile stages of rainbow trout ( Oncorhynchus mykiss)

The present study investigates the effects of dietary phospholipids (PL) on intestinal lipid absorption in fry and early juvenile stages of rainbow trout ( Oncorhynchus mykiss) fed fresh or oxidized lipids. Six semi-purified casein-based diets were formulated with 12% fresh or oxidized fish oil and 6% PL supplied as soybean lecithin or egg lecithin or 6% soybean oil for PL-free diets. Each diet was distributed to 3 replicate tanks of 700 swim-up fry (initial mean body weight: 66 mg) or 200 early juvenile fish (initial mean body weight: 455 mg) over a 3-week or 17-week growth trial at a water temperature of 17 °C. Feeding oxidized compared to fresh lipids significantly reduced body weight of both fry and juvenile trout (0.15 ± 0.02 vs. 0.28 ± 0.07 g and 10 ± 2 vs. 22 ± 2 g, respectively). At fry stages, within groups fed fresh lipids, trout fry fed the PL-supplemented diets had a significantly higher final body weight than fry fed the PL-free diet (0.31 ± 0.06 vs. 0.21 ± 0.02 g). Histological examination revealed a higher occurrence of intestinal steatosis (accumulation of large lipid droplets in the enterocytes of the anterior intestine) in fish fed the PL-free diet compared to fish fed PL-supplemented diets at fry stages (13/16 vs. 1/32), whereas no significant differences were recorded at juvenile stages (1/18 vs. 0/18). The levels of circulating total cholesterol, triacylglycerol (TAG) and PL in the juvenile fish were not significantly modified by dietary PL supplementation whereas the plasma TAG/PL ratio was lower in juvenile fish fed oxidized compared to fresh lipids (0.31 ± 0.06 vs. 0.45 ± 0.08). At the fry stages, within groups fed fresh lipids, expression of the gene coding for apolipoprotein B (apoB) was 2.3-fold reduced in the intestine of fish fed the PL-free diet compared to the PL-supplemented diets whereas no significant difference was recorded at juvenile stages. The expression of other apolipoproteins or key-enzymes involved in intestinal lipoprotein synthesis was not significantly modified by dietary PL supplementation. In conclusion, the results indicate that the beneficial effect of dietary PL on growth and intestinal lipid absorption is restricted to the fry stages and support previous opinions that PL are needed for intestinal TAG absorption. The limiting step seems to be the synthesis of apoB by the enterocytes in rainbow trout fry fed fresh lipids but this requires further investigation in fish fed oxidized lipids.

The glucagon-like peptide 1 receptor is essential for postprandial lipoprotein synthesis and secretion in hamsters and mice

Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors attenuate postprandial lipaemia through mechanisms that remain unclear. As dyslipidaemia is a contributing risk factor for cardiovascular disease in type 2 diabetes, we examined the mechanisms linking pharmacological and physiological regulation of GLP-1 action to control of postprandial lipid metabolism. Postprandial lipid synthesis and secretion were assessed in normal and fructose-fed hamsters and in wild-type mice that were treated with or without sitagliptin. Apolipoprotein B-48 (ApoB-48) synthesis and secretion were also examined in primary enterocyte cultures. The importance of exogenous vs endogenous GLP-1R signalling for regulation of intestinal lipoprotein synthesis and secretion was assessed in mice and hamsters treated with the GLP-1R agonist exendin-4, the GLP-1R antagonist exendin(9-39) and in Glp1r (+/+) vs Glp1r (-/-) mice. Sitagliptin decreased fasting plasma triacylglycerol, predominantly in the VLDL fraction, as well as postprandial triacylglycerol-rich lipoprotein (TRL)-triacylglycerol, TRL-cholesterol and TRL-ApoB-48 in hamsters and mice. GLP-1R activation with exendin-4 alone also decreased plasma and TRL-ApoB-48 in hamsters and mice, and reduced secretion of ApoB-48 in hamster enterocyte cultures. Conversely, blockade of endogenous GLP-1R signalling by the antagonist exendin(9-39) or genetic elimination of GLP-1R signalling in Glp1r (-/-) mice enhanced TRL-ApoB-48 secretion in vivo. Co-administration of exendin(9-39) also abolished the hypolipidaemic effect of sitagliptin. Potentiation of endogenous incretin action via DPP-4 inhibition or pharmacological augmentation of GLP-1R signalling reduces intestinal secretion of triacylglycerol, cholesterol and ApoB-48. Moreover, endogenous GLP-1R signalling is essential for the control of intestinal lipoprotein biosynthesis and secretion.

Diet-Induced Changes in Liver and Bile but Not Brain Fatty Acids Can Be Predicted from Differences in Plasma Phospholipid Fatty Acids in Formula- and Milk-Fed Piglets ,

The fatty acid composition of plasma phospholipids differs between infants fed formula and infants fed human milk, but the extent to which this is accompanied by differences in tissue phospholipid fatty acids is unclear. This paper describes analysis of plasma, liver and brain fatty acids from piglets fed one of seven formulas, varying in saturated, monounsaturated, (n-6) and (n-3) fatty acids or sow milk from birth for 18 d. Bile fatty acids were analyzed because they are secreted from liver and may be an important source of fatty acids for intestinal lipoprotein synthesis. The results were used to determine the relation between diet-related differences in plasma phospholipid fatty acids and those in brain, liver and bile. Where significant associations were found, prediction limits were constructed to assess the usefulness of analysis of plasma phospholipid fatty acids to predict diet-induced changes in tissue fatty acids. The proportions (g/100 g fatty acids) of 16:0, 18:0, 18:1, 18:2(n-6) and 20:4(n-6) in plasma phospholipids were significantly associated with the proportions of the same fatty acids in liver and bile, but not brain. The results show a reasonably precise, predictable association between plasma and liver, and plasma and bile fatty acids. Brain 20:4(n-6) and 22:6(n-3), in contrast, were not reliably associated with plasma phospholipid 20:4(n-6) and 22:6(n-3) for piglets fed milk or formula providing about 1.5% energy as 18:3(n-3).

Caco-2 cells as a model for intestinal lipoprotein synthesis and secretion.

Caco-2 cells, an intestinal cell line derived from a human colorectal carcinoma that spontaneously differentiates under standard culture conditions, lends itself to the in vitro study of human gut in view of its efficient intestinal transport processes. Among its multiple biological functions are those related to the absorption, transport, and metabolism of lipids and lipoproteins. Despite their intestinal origin, confluent Caco-2 cell monolayers primarily express L-FABP for the uptake of apical dietary long chain fatty acids, incorporating them into triglycerides by the glycerol 3-phosphate pathway, and assembling very-low-density lipoprotein, high-density lipoprotein, and low-density lipoprotein. The monoacyl-glycerol pathway is inactive in Caco-2 cells. Furthermore, the secretion of newly synthesized triglyceride-rich lipoproteins is very restricted, despite abundant production of apolipoprotein (apo) B. The regulation of apoB synthesis and its mRNA editing at the enterocyte level has been intensively examined in Caco-2 cells. Luminal fatty acids, calcium ion, as well as vitamins and hormones are known to modulate the apoB-48/apoB-100 at the transcriptional and/or translational level. The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and acyl-CoA: (cholesterol acyltransferase), the key enzymes governing intracellular cholesterol handling, have also been extensively examined in Caco-2 cells. In many respects this cell line provides an excellent in vitro model for the investigation of intestinal lipoprotein metabolism; however, their limited secretion capacity remains a potential drawback to comparisons with the in vivo physiological state.

The ontogeny and site of intestinal lipid and lipoprotein synthesis.

The developmental aspects of characteristic intestinal lipoprotein synthesis, chlomicrons (CM), very low density lipoproteins (VLDL) and high density lipoproteins (HDL), are unknown. Our objective was to determine the ontogeny of intestinal lipid and lipoprotein synthesis in both the jejunum and the ileum. Explants of the jejunum and the ileum from fetal (F) (18-19 days of gestation), suckling (S) (5 days old) and weaning (W) (23 days old) rats were cultured in the presence of [14C]-oleic acid to examine lipid synthesis. The results indicate the following. (1) The incorporation of oleic acid is higher for the fetal explants. However, the efficiency of esterification of free fatty acids (FFA) into triglycerides (TG) in the jejunum increases with age (33% F, 37% S, 48% W) (P less than 0.05, by ANOVA). (2) The same profile is found at the ideal site for the incorporation of oleic acid. However, the capacity for the synthesis of TG is more intense at the suckling period (34% F, 54% S, 42% W) (P less than 0.05, by ANOVA). (3) The relative content of TG in CM changes with age: F, 90-93%; S, 80-84%; W, 33-40%. (4) A low percentage of TG content is found in CM at the weaning period while high levels are detectable in VLDL (40-42%). (5) A most significant difference is noted between the jejunum and the ileum in TG synthesis at the suckling period. The ileum synthesizes 53% more TG than the jejunum (P less than 0.025). (6) HDL particles contain substantial amounts of FFA. Nevertheless, they were also found to be able to transport TG mainly in the suckling rat. Thus, this study demonstrates that with growth the rat is able to synthesize CM, VLDL and HDL. Our findings indicate changes in the synthesis of intestinal lipids and lipoproteins, depending on both the development and the site, which suggests an ontogeny. These modifications can be attributed to dietary and hormonal influences present during the period of development.

Intestinal lipoprotein synthesis in control and hypercholesterolemic rats

Previous studies in our laboratory have shown that very-low-density lipoproteins (VLDL) synthesized by the intestine of the diet-induced hypercholesterolemic rat are enriched in cholesteryl esters and unesterified cholesterol compared with intestinal VLDL from control rats. In these studies, we isolated and characterized nascent intestinal Golgi intermediate-density lipoproteins (IDL, d 1.006–1.040 g/ ml) and studied isotope incorporation into apolipoproteins of Golgi VLDL from control and hypercholesterolemic rats. IDL were triacylglycerol-rich lipoproteins but contained more cholesteryl ester and protein than the corresponding Golgi VLDL fractions. IDL from hypercholesterolemic rats were enriched in cholesteryl esters to a greater extent than IDL from control rats. The apolipoprotein patterns of IDL fractions were the same as those of intestinal Golgi VLDL, consisting of apolipoproteins (apo) B-48, A-I and A-IV. Time-course isotope incorporation curves for apo A-I and A-IV in Golgi VLDL were similar, but they differed from curves for apo B-48. None of these curves was markedly altered in the hypercholesterolemic rat. We conclude that the major effect of increased dietary cholesterol on intestinal lipoprotein biosynthesis is to increase the percentage of cholesteryl esters in Golgi lipoproteins. Dietary cholesterol does not alter the apolipoprotein composition of Golgi lipoproteins, nor does it have a significant effect on the pattern of isotope incorporation into apolipoproteins of Golgi VLDL. The effect of cholesteryl ester enrichment on the subsequent metabolism of these particles in the circulation and the effect of these particles on hepatic lipoprotein production remain to be determined.

A novel cell line (Caco-2) for the study of intestinal lipoprotein synthesis.

Lipoprotein synthesis by the colonic adenocarcinoma cell line Caco-2 was investigated to assess the utility of this cell line as a model for the in vitro study of human intestinal lipid metabolism. Electron micrographic analysis of conditioned medium revealed that under basal conditions of culture post-confluent Caco-2 cells synthesize and secrete lipoprotein particles. Lipoproteins of density (d) less than 1.063 g/ml consist of a heterogeneous population of particles (diameter from 10 to 90 nm). This fraction consists of very low density lipoproteins (d less than 1.006 g/ml) and low density lipoproteins (d = 1.019-1.063 g/ml). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled Caco-2 lipoproteins revealed that very low density lipoproteins contain apolipoprotein E (apoE) and C apolipoproteins, while low density lipoproteins contained apoB-100, apoE, apoA-I, and C apolipoproteins. The 1.063-1.21 g/ml density fraction contained two morphological entities, discoidal (diameter 15.6 +/- 3.9 nm) and round high density lipoprotein particles (diameter 10.2 +/- 2.3 nm). The high density lipoproteins contained apoA-I, apoB-100, apoB-48, apoE, and the C apolipoproteins. Using isoelectric focusing polyacrylamide gel electrophoresis newly secreted apoA-I was identified as pro-apoA-I. ApoE and apoC-III released by Caco-2 cells were highly sialylated. mRNA species for apoA-I, apoC-III, and apoE, but not apoA-IV were identified by Northern blot analysis. ApoA-I, apoB, and apoE were visualized in Caco-2 cells by immunolocalization analysis. This intestinal cell line may be useful for in vitro studies of nutritional and hormonal regulation of lipoprotein synthesis.

Evidence for separate pathways of chylomicron and very low-density lipoprotein assembly and transport by rat small intestine.

Previously, we demonstrated that the hydrophobic surfactant Pluronic L-81 (L-81) inhibits the intestinal formation and transport of chylomicrons (CM) but not of very low-density lipoprotein-sized (VLDL) particles. The present study was undertaken to determine whether infusion of egg lecithin results mainly in secretion of VLDL by the small intestine and whether L-81 has any effect on their formation and secretion. Intestinal fistula rats were infused intraduodenally at a rate of 3 ml/h with a lipid emulsion containing 20 mM egg lecithin and 19 mM sodium taurocholate for 8 h. This was then followed by another 8 h of infusion of a similar lipid emulsion but with 0.5 mg/h of L-81 added. Lymphatic lipid output was measured, and lymph lipoproteins were sized by use of electron microscopy. Whether L-81 was present or not, no significant difference was detected in the lymphatic triglyceride, phospholipid, or cholesterol outputs. Based on agarose gel electrophoresis, sizing of intestinal lymph lipoproteins, and also the determination of lipid in the intestinal lymph CM and VLDL as separated by ultracentrifugation, VLDL were the major lipoproteins present in lymph during the infusion of egg lecithin. Thus, intraduodenal infusion of egg lecithin in the rat results mainly in the transport of VLDL and is not affected by the administration of L-81. The results suggest that CM and VLDL are assembled separately by the enterocytes and indicate the usefulness of L-81 in further investigating the pathways and regulation of intestinal lipoprotein synthesis, assembly, and secretion.

Intestinal lipoprotein synthesis. Comparison of nascent Golgi lipoproteins from chow-fed and hypercholesterolemic rats.

Hypercholesterolemia, induced by a cholesterol-enriched diet, is associated with distinctive modifications in the serum lipoproteins of a variety of species. Present in the serum of these animals are several classes of lipoproteins enriched in cholesteryl esters and apolipoprotein E. To investigate the role of intestinal lipoprotein synthesis in diet-induced hypercholesterolemia, we characterized nascent lipoproteins retrieved from Golgi apparatus-rich fractions of intestinal epithelial cells from chow-fed control and hypercholesterolemic rats. To eliminate chylomicrons from the preparations, rats were fasted overnight prior to the experiments. Golgi very low density lipoproteins (d less than 1.006 g/ml) from control rats were triglyceride-rich lipoproteins that migrated slightly slower than pre-beta migrating serum very low density lipoproteins. These particles contained apoproteins B-240, A-IV, and A-I. Golgi very low density lipoproteins from hypercholesterolemic rats were likewise triglyceride-rich lipoproteins migrating electrophoretically like control Golgi very low density lipoproteins and they contained apoproteins B-240, A-IV, and A-I. However, these latter particles contained less triglyceride and more cholesterol compared to control Golgi very low density lipoproteins. In addition, by radioisotope incorporation studies, Golgi very low density lipoproteins from hypercholesterolemic rats contained relatively more apoprotein A-IV (21.6 vs. 11.0%) and less apoprotein B-240 (17.0 vs. 27.0%) than found in control Golgi very low density lipoproteins. Approximately 60% of the total apoprotein radioactivity was found in apoprotein A-I in both preparations. We conclude that intestinal lipoprotein synthesis is modified by diet-induced hypercholesterolemia. The significance of these modifications with respect to the marked hypercholesterolemia observed in these animals remains to be determined.