Lymph Lipids Research Articles

Lymphatic Uptake of a Highly Lipophilic Protease Inhibitor Prodrug from a Lipid-Based Formulation is Limited by Instability in the Intestine

Dabigatran etexilate (DABE) is a lipophilic double alkyl ester prodrug of dabigatran (DAB) which is a serine protease inhibitor used clinically as an anticoagulant. Recently, translocation of serine protease enzymes, including trypsin, from the gut into the mesenteric lymph and then blood has been associated with organ failure in acute and critical illnesses (ACIs). Delivery of DABE into mesenteric lymph may thus be an effective strategy to prevent organ failure in ACIs. Most drugs access the mesenteric lymph in low quantities following oral administration, as they are rapidly transported away from the intestine via the blood. Here, we examine the potential to deliver DABE into the mesenteric lymph by promoting association with lymph lipid transport pathways via co-administration with a lipid-based formulation (LBF). A series of self-emulsifying LBFs were designed and tested in vitro for their potential to form stable DABE loaded emulsions and keep DABE solubilised and stable over time in simulated gastrointestinal conditions. The LBFs were found to form fine emulsions with a droplet size of 214 ± 30 nm and DABE was stable in the formulation. The stability of DABE in vitro in simulated intestinal conditions, plasma and lymph samples was also evaluated to ensure stability in collected samples and to evaluate whether the prodrug is likely to release active DAB. Ultimately, a highly uniform and stable self-emulsifying Type III A LBF of DABE was chosen for progression into in vivo studies in male Sprague Dawley rats to confirm the lymphatic uptake and plasma pharmacokinetics. Both in vitro and in vivo in plasma and lymph, DABE was rapidly converted to an intermediate and DAB. The main species present in vivo in both plasma and lymph was DAB and mass transport of DABE and DAB in lymph was minimal (∼0.5 % of dose). Importantly, the concentration of DABE in lymph was substantially (20–176 fold) higher than in plasma, supporting that if the prodrug were stable and did not convert to DAB in the intestine, it would be lymphatically transported. Future studies will therefore focus on optimizing the design of the prodrug and formulation to improve stability during absorption and further promote lymphatic uptake.

Release of Lipids Stored in the Intestine by Glucagon-Like Peptide-2 Involves a Gut-Brain Neural Pathway.

The gut hormone GLP-2 (glucagon-like peptide-2) plays important roles in lipid handling in the intestine. During postabsorptive stage, it releases preformed chylomicrons stored in the intestine, the underlying mechanisms of which are not well understood. Previous studies implicate the involvement of neural pathways in GLP-2's actions on lipid absorption in the intestine, but the role of such mechanisms in releasing postabsorptive lipid storage has not been established. Here, in mesenteric lymph duct cannulated rats, we directly tested whether gut-brain neural communication mediates GLP-2's effects on postabsorptive lipid mobilization in the intestine. We performed total subdiaphragmatic vagotomy to disrupt the gut-brain neural communication and analyzed lipid output 5 hours after a lipid load in response to intraperitoneal GLP-2 or saline. Peripheral GLP-2 administration led to increased lymph lipid output and activation of proopiomelanocortin neurons in the arcuate nucleus of hypothalamus. Disruption of gut-brain neural communication via vagotomy blunted GLP-2's effects on promoting lipid release in the intestine. These results, for the first time, demonstrate a novel mechanism in which postabsorptive mobilization of intestinal lipid storage by GLP-2 enlists a gut-brain neural pathway.

Increasing Linker Chain Length and Intestinal Stability Enhances Lymphatic Transport and Lymph Node Exposure of Triglyceride Mimetic Prodrugs of a Model Immunomodulator Mycophenolic Acid.

Targeted delivery of immunomodulators to the lymphatic system has the potential to enhance therapeutic efficacy by increasing colocalization of drugs with immune targets such as lymphocytes. A triglyceride (TG)-mimetic prodrug strategy has been recently shown to enhance the lymphatic delivery of a model immunomodulator, mycophenolic acid (MPA), via incorporation into the intestinal TG deacylation-reacylation and lymph lipoprotein transport pathways. In the current study, a series of structurally related TG prodrugs of MPA were examined to optimize structure-lymphatic transport relationships for lymph-directing lipid-mimetic prodrugs. MPA was conjugated to the sn-2 position of the glyceride backbone of the prodrugs using linkers of different chain length (5-21 carbons) and the effect of methyl substitutions at the alpha and/or beta carbons to the glyceride end of the linker was examined. Lymphatic transport was assessed in mesenteric lymph duct cannulated rats, and drug exposure in lymph nodes was examined following oral administration to mice. Prodrug stability in simulated intestinal digestive fluid was also evaluated. Prodrugs with straight chain linkers were relatively unstable in simulated intestinal fluid; however, co-administration of lipase inhibitors (JZL184 and orlistat) was able to reduce instability and increase lymphatic transport (2-fold for a prodrug with a 6-carbon spacer, i.e., MPA-C6-TG). Methyl substitutions to the chain resulted in similar trends in improving intestinal stability and lymphatic transport. Medium- to long-chain spacers (C12, C15) between MPA and the glyceride backbone were most effective in promoting lymphatic transport, consistent with increases in lipophilicity. In contrast, short-chain (C6-C10) linkers appeared to be too unstable in the intestine and insufficiently lipophilic to associate with lymph lipid transport pathways, while very long-chain (C18, C21) linkers were also not preferred, likely as a result of increases in molecular weight reducing solubility or permeability. In addition to more effectively promoting drug transport into mesenteric lymph, TG-mimetic prodrugs based on a C12 linker resulted in marked increases (>40 fold) in the exposure of MPA in the mesenteric lymph nodes in mice when compared to administration of MPA alone, suggesting that optimizing prodrug design has the potential to provide benefit in targeting and modulating immune cells.

Intestinal Synthesis and Lymphatic Secretion of Apolipoprotein A–IV Vary with Chain Length of Intestinally Infused Fatty Acids in Rats

To evaluate the hypothesis that stimulation of intestinal apolipoprotein (apo) A–IV by dietary fat depends upon assembly and transport of chylomicrons, we examined the effect of duodenal infusion of fatty acids of graded chain length on mucosal synthesis and lymphatic output of lipid and apo A–IV. Rats with duodenal cannulas and mesenteric lymph fistulas were given 8-h duodenal infusions of lipid emulsions containing either butyric (4:0), caprylic (8:0), lauric (12:0), myristic (14:0), stearic (18:0), oleic (18:1), linoleic (18:2) or arachidonic (20:4) acids, or tributyrin, tricaprylin or triolein. Lymph outputs of triglyceride, phospholipid and apo A–IV were measured at 0, 2, 4, 5, 6, 7 and 8 h after the start of lipid infusion. Significant increases in lymph lipid (triglyceride, phospholipid) and apo A–IV output were observed in response to long-chain fatty acids (14:0, 18:0, 18:1, 18:2, 20:4) or triolein; short- or medium-chain fatty acids (4:0, 8:0, 12:0) or tributyrin or tricaprylin produced no significant increase in lymph lipid output above basal levels. Similarly, increased jejunal mucosal synthesis of apo A–IV was observed in response to duodenal infusion of oleic acid but not butyric or caprylic acid. These results provide direct support for the hypothesis that stimulation of apo A–IV by dietary fat depends upon transport of absorbed lipid via chylomicrons in lymph.

Impact of Sequential Lipid Meals on Lymphatic Lipid Absorption and Transport in Rats.

The sequential meal pattern has recently received more attention because it reflects a phycological diet style for human beings. The present study investigated the effects of the second lipid meal on lymphatic lipid absorption and transport in adult rats following a previous lipid meal. Using the well-established lymph fistula model, we found that the second lipid meal significantly increased the lymphatic output of triglycerides, cholesterol, phospholipids, and non-esterified fatty acids compared with a single lipid meal. Besides that, the time reaching the peak of each lipid output was significantly faster compared with the first lipid meal. Additionally, the second lipid meal significantly increased the lymphatic output of apolipoprotein A-IV (ApoA-IV), but not apolipoprotein B-48 (ApoB-48) or apolipoprotein A-I (ApoA-I). Interestingly, the triglyceride/apoB-48 ratio was significantly increased after the second lipid meal, indicating the increased chylomicron size in the lymph. Finally, the second lipid meal increased the lymphatic output of rat mucosal mast cell protease II (RMCPII). No change was found in the expression of genes related to the permeability of lymphatic lacteals, including vascular endothelial growth factor-A (Vegfa), vascular endothelial growth factor receptor 1 (Flt1), and Neuropilin1 (Nrp1). Collectively, the second lipid meal led to the rapid appearance of bigger-sized chylomicrons in the lymph. It also increased the lymphatic output of various lipids and apoA-IV, and mucosal mast cell activity in the intestine.

Dietary lysophospholipids reduce lymphatic cholesterol transport compared with dietary phospholipids in thoracic lymph-duct cannulated rats.

Dietary phospholipids have been traditionally known to affect micelle formation. Egg yolk-derived lysophospholipids (LysoPL) are commercially available. We investigated the effects of dietary LysoPL on lymphatic lipid transport. We also compared sn-1 LysoPL and sn-2 LysoPL, which have different fatty acyl esterification positions. Thoracic lymph duct-cannulated rats were fed a diet supplemented with egg yolk-derived sn-1 LysoPL, sn-2 LysoPL, or phospholipids (PL). The amount of lymphatic lipid transport was also evaluated. Time courses of transport were applied to the one-compartment model as one of the pharmacokinetic analyses. The solubility of cholesterol in bile acid micelles was measured. Compared to the PL diet, the sn-1 and sn-2 LysoPL diets significantly reduced the lymphatic transport of cholesterol. There were no differences in the lymphatic PL and TAG transport. There was no difference in cholesterol transport between the sn-1 LysoPL group and the sn-2 LysoPL group; however, the transport rate constant at a decrease in lymphatic cholesterol was lower in the sn-1 LysoPL group than in the sn-2 LysoPL group. Cholesterol solubility in bile acid micelles was significantly decreased in the sn-1 LysoPL and sn-2 LysoPL groups compared to that in the PL group. Dietary LysoPL affects the behavior of intestinal cholesterol and suppresses lymphatic cholesterol transport.

Visceral obesity and insulin resistance associate with CD36 deletion in lymphatic endothelial cells

Disruption of lymphatic lipid transport is linked to obesity and type 2 diabetes (T2D), but regulation of lymphatic vessel function and its link to disease remain unclear. Here we show that intestinal lymphatic endothelial cells (LECs) have an increasing CD36 expression from lymphatic capillaries (lacteals) to collecting vessels, and that LEC CD36 regulates lymphatic integrity and optimizes lipid transport. Inducible deletion of CD36 in LECs in adult mice (Cd36ΔLEC) increases discontinuity of LEC VE-cadherin junctions in lacteals and collecting vessels. Cd36ΔLEC mice display slower transport of absorbed lipid, more permeable mesenteric lymphatics, accumulation of inflamed visceral fat and impaired glucose disposal. CD36 silencing in cultured LECs suppresses cell respiration, reduces VEGF-C-mediated VEGFR2/AKT phosphorylation and destabilizes VE-cadherin junctions. Thus, LEC CD36 optimizes lymphatic junctions and integrity of lymphatic lipid transport, and its loss in mice causes lymph leakage, visceral adiposity and glucose intolerance, phenotypes that increase risk of T2D.

Rapeseed Lecithin Increases Lymphatic Lipid Output and α-Linolenic Acid Bioavailability in Rats

Soybean lecithin, a plant-based emulsifier widely used in food, is capable of modulating postprandial lipid metabolism. With arising concerns of sustainability, alternative sources of vegetal lecithin are urgently needed, and their metabolic effects must be characterized. We evaluated the impact of increasing doses of rapeseed lecithin (RL), rich in essential α-linolenic acid (ALA), on postprandial lipid metabolism and ALA bioavailability in lymph-cannulated rats. Male Wistar rats (8 weeks old) undergoing a mesenteric lymph duct cannulation were intragastrically administered 1 gof an oil mixture containing 4% ALA and 0, 1, 3, 10, or 30% RL (5 groups). Lymph fractions were collected for 6 h.Lymphlipidsandchylomicrons (CMs) were characterized. The expression of genes implicated in intestinal lipid metabolism was determined in the duodenum at 6 h.Datawasanalyzedusing either sigmoidal or linear mixed-effects models, or one-way ANOVA, where appropriate. RL dose-dependently increased the lymphatic recovery (AUC) of total lipids (1100 μg/mL·hperadditional RL%; P=0.010) and ALA (50 μg/mL·hperadditional RL%; P=0.0076). RL induced a faster appearance of ALA in lymph, as evidenced by the exponential decrease of the rate of appearance of ALA with RL (R2=0.26; P=0.0064). Although the number of CMs was unaffected by RL, CM diameter was increased in the 30%-RL group, compared to the control group (0% RL), by 86% at 3-4 h(P=0.065) and by 81% at 4-6 h(P=0.0002) following administration. This increase was positively correlated with the duodenal mRNA expression of microsomal triglyceride transfer protein (Mttp; ρ= 0.63; P=0.0052). The expression of Mttp and secretion-associated, ras-related GTPase 1 gene homolog B (Sar1b, CM secretion), carnitine palmitoyltransferase IA (Cpt1a) and acyl-coenzyme A oxidase 1 (Acox1, beta-oxidation), and fatty acid desaturase 2 (Fads2, bioconversion of ALA into long-chain n-3 PUFAs) were, respectively, 49%, 29%, 74%, 48%, and 55% higher in the 30%-RL group vs. the control group (P<0.05). In rats, RL enhanced lymphatic lipid output, as well as the rate of appearance of ALA, which may promote its subsequent bioavailability and metabolic fate.

Differential Metabolic Impact of Natural Food-Grade Emulsifiers Rich in Alpha-Linolenic Acid

Abstract Objectives Dietary synthetic emulsifiers have recently been shown to promote metabolic syndrome and considerably alter gut microbiota. Conversely, natural emulsifiers such as milk polar lipids (PL) are associated with beneficial metabolic effects. The effects of plant PL remain, however, poorly described. Our objective was to evaluate, using two complimentary rodent models, the impact of nutritional doses of lecithin (≤10%) of vegetal sources alternative to soy on gut microbiota, postprandial lipid metabolism, and the bioavailability of an essential plant lipid, alpha-linolenic acid (ALA). Methods For 5 days, male Swiss mice (n = 60) were fed normolipidic diets (identical ALA content) containing 0, 1, 3 or 10% rapeseed lecithin (RL) or 10% soy lecithin. Following an overnight fast, the mice were force-fed the same oil mix and euthanised after 90 min. As a mechanistic study, male Wistar rats (n = 30) with mesenteric duct cannulation were fed 5 oil mixtures containing 0 to 20% RL. Lymph fractions were collected up to 6 h post-gavage. Plasma and lymph lipid composition was determined using GC-FID, chylomicron (CM) size using light-scattering spectroscopy, and intestinal gene expression and faecal microbiota composition by RT-qPCR. Results In mice, the consumption of lecithin significantly increased levels of faecal Clostridium leptum (P &amp;lt; 0.001), regardless of lecithin origin or dose. The partial replacement of oil with lecithin did not significantly alter plasma total lipids nor the expression of genes of intestinal lipid absorption. The percentage of ALA in plasma triglycerides was significantly higher in the 10% RL group compared to other groups (P &amp;lt; 0.05). In rats, RL significantly and dose-dependently increased the rate of appearance (P &amp;lt; 0.01) and concentration of ALA in lymph (P &amp;lt; 0.01). 20% RL additionally increased CM size and expression of genes of CM secretion (Mttp, Sar1b; P &amp;lt; 0.05). Conclusions Our results reveal that, whereas both rapeseed and soy lecithin improved gut microbiota composition by increasing the anti-inflammatory Clostridium leptum bacterial group, only rapeseed lecithin enhanced ALA bioavailability. This study illustrates the importance of considering the use of natural emulsifiers, especially rapeseed lecithin, as plant-based food ingredients with potential health benefits. Funding Sources ANRT and UMT ACTIA BALI.

Intestinal Lymph Flow, and Lipid and Drug Transport Scale Allometrically From Pre-clinical Species to Humans.

The intestinal lymphatic system transports fluid, immune cells, dietary lipids, and highly lipophilic drugs from the intestine to the systemic circulation. These transport functions are important to health and when dysregulated contribute to pathology. This has generated significant interest in approaches to deliver drugs to the lymphatics. Most of the current understanding of intestinal lymph flow, and lymphatic lipid and drug transport rates, comes from in vitro studies and in vivo animal studies. In contrast, intestinal lymphatic transport studies in human subjects have been limited. Recently, three surgical patients had cannulation of the thoracic lymph duct for collection of lymph before and during a stepwise increase in enteral feed rate. We compared these data to studies where we previously enterally administered controlled quantities of lipid and the lipophilic drug halofantrine to mice, rats and dogs and collected lymph and blood (plasma). The collected lymph was analyzed to compare lymph flow rate, triglyceride (TG) and drug transport rates, and plasma was analyzed for drug concentrations, as a function of enteral lipid dose across species. Lymph flow rate, TG and drug transport increased with lipid administration in all species tested, and scaled allometrically according to the equation A = aME where A is the lymph transport parameter, M is animal body mass, a is constant and E is the allometric exponent. For lymph flow rate and TG transport, the allometric exponents were 0.84–0.94 and 0.80–0.96, respectively. Accordingly, weight normalized lymph flow and TG mass transport were generally lower in larger compared to smaller species. In comparison, mass transport of drug via lymph increased in a greater than proportional manner with species body mass with an exponent of ∼1.3. The supra-proportional increase in lymphatic drug transport with species body mass appeared to be due to increased partitioning of drug into lymph rather than blood following absorption. Overall, this study proposes that intestinal lymphatic flow, and lymphatic lipid and drug transport in humans is most similar to species with higher body mass such as dogs and underestimated by studies in rodents. Notably, lymph flow and lipid transport in humans can be predicted from animal data via allometric scaling suggesting the potential for similar relationships with drug transport.

Intestinal lymphatic vessels and their role in chylomicron absorption and lipid homeostasis.

In this review, we describe novel findings related to intestinal lipid transport in lymphatic vessels. Studies have shown that chylomicron entry to lacteals and lymph movement in intestinal lymphatic capillaries is an active process. Regulators of this intestinal chylomicron transport include among others the autonomous nervous system, transcription factors like PLAGL2, and molecular regulators, such as VEGF-A/Nrp1/VEGFR1, VEGF-C/VEGFR3, DLL4, CALCRL and GLP-2. Chylomicron transport in intestinal lymphatics is now emerging not only as an option for drug delivery but also as a new candidate for drug targeting in lipid-related disorders. Dysfunctions of lymphatic lipid transport can result in conditions such as dyslipidaemia. Intestinal lymphatics also provide several potential therapeutic possibilities: molecular regulation of lacteal cell-to-cell junctioning and lymph flow could provide new ways of treating conditions like hyperlipidaemia and associated diseases, such as atherosclerosis and other cardiovascular diseases, obesity, diabetes and fatty-liver disease. The intestinal lymphatic system can also be employed to deliver lipid nanoparticles as drug carriers to the venous circulation for improved treatment outcome. These findings highlight the importance and need for research on the different players of intestinal lymphatics in dietary lipid handling and therapeutic applications.

In vitro lipolysis and lymphatic absorption of n-3 long-chain PUFA in the rat: influence of the molecular lipid species as carrier.

The aim of this work was to study the bioavailability of fatty acids (FA), focusing on n-3 long-chain (LC) PUFA, carried by different molecular lipid species, that is, phospholipids (PL) or TAG, with three formulations based on fish oils or marine PL, providing a similar n-3 LC PUFA amount. The digestive lipolysis was first assessed using an in vitro enzymatic model. Then, intestinal absorption and enterocyte metabolism were investigated in vivo, on male Wistar rats through lymph lipid analysis. The in vitro results showed that the release of n-3 LC PUFA from lipolysis was increased by 48% when FA were provided as PL rather than TAG. The in vivo results demonstrated that EPA and DHA from both TAG and PL were similarly absorbed and incorporated into lymph lipids. However, DHA was mainly distributed at the sn-1/3 positions of lymph TAG when provided as marine PL, whereas it was equally distributed at the three positions with marine TAG. On the whole, even if the molecular lipid species of n-3 LC PUFA did not greatly modify the in vivo digestion and absorption steps, it modulated the rearrangement of DHA on the glyceride positions of the lymph TAG, which may further impact the DHA metabolic fate and tissue accretion. Consequently, the present study has provided data which may be used to formulate lipid diets rich in DHA in the context of an insufficient consumption of n-3 PUFA in Western countries.

Enhancement of rat lymphatic lipid transport by glucose or amino acids ingestion.

To elucidate the effect of simultaneously fed carbohydrate or protein on lipid absorption, lymphatic lipid transports in the rat intestine were observed with or without simultaneous feeding of glucose or amino acids. A lipid emulsion containing 40 μmol/h (35.4 mg/h) of triolein, 2.74 μmol/h (1.06 mg/h) of cholesterol, 7.8 μmol/h (6.08 mg/h) of egg phosphatidylcholine without any additive (the Control group), with 560 mmol/h (300 mg/h) of glucose (the Glucose group), or with 400 mmol/h (150 mg/h) of amino acids (the Amino Acids group), was infused intraduodenally at 3 mL/h for 8 h into mesenteric lymph‐fistula rats. The amounts of triglyceride transported in lymph for 8 h were 185 ± 12 (mean ± SE) mg in the Amino Acids group (n = 4), 175 ± 3 mg in the Glucose group (n = 5), and 147 ± 7 mg in the Control group (n = 4), respectively, with a statistically significant difference (P < 0.05) among the groups. The amounts of cholesterol transported in lymph for 8 h of the Amino Acid group and the Glucose group seemed to be larger than that of the Control group. The amount of phosphatidylcholine transported in lymph for 8 h were 16.4 ± 1.0 mg in the Amino Acids group, 15.7 ± 0.4 mg in the Glucose group, and 12.4 ± 0.3 mg in the Control group, respectively, with a statistically significant difference (P < 0.01) among the groups. Simultaneous glucose or amino acids feeding enhanced lymphatic lipid transport in the rat intestine during lipid feeding.

Alleviation of Metabolic Syndrome with Dietary Egg White Protein.

Abdominal fat accumulation causes metabolic syndrome, which is a cluster of metabolic abnormalities such as dyslipidemia, glucose intolerance, insulin resistance or hyperinsulinemia, and hypertension, leading to the development of diabetes and cardiovascular disease. Diets are known to contribute to the development or prevention of metabolic syndrome. Several studies have reported that the quality of dietary proteins may be an important modulator of the risk of this syndrome. We investigated the effects of consuming egg white protein (EWP) or lactic-fermented egg white (LE), an easy-to-consume form of egg white, on the development of metabolic syndrome in animal models and humans. In comparison with casein, dietary EWP decreased lymphatic lipid transport in thoracic lymph duct-cannulated rats. In an in vitro experiment, EWP pepsin hydrolysate decreased the cholesterol micellar solubility and cholesterol transfer rate from micelles to oil phase, and increased water-holding capacity, settling volume in water, and relative viscosity compared with casein pepsin hydrolysate. The daily consumption of LE for 8 weeks reduced serum total cholesterol and LDL cholesterol levels in men with mild hypercholesterolemia. Furthermore, dietary EWP reduced the body fat mass of rats by increasing the body protein mass and accelerating hepatic β-oxidation. The daily consumption of LE for 12 weeks reduced the visceral fat area and improved the ratio of the visceral to subcutaneous fat area. Taken together, these results indicated that dietary EWP and LE would be useful for preventing or alleviating metabolic syndrome.

Abstract 552: Enhancement of Rat Lymphatic Lipid Transport by Glucose or Amino Acids Ingestion

Objectives: Dietary lipid absorption usually occurs with absorption of other nutrients because lipid is contained in foods fed as a meal. The effect of simultaneously fed carbohydrate and protein on lipid absorption is not clear. In this study, lymphatic lipid transports in the rat intestine were observed during intraduodenal lipid feeding with or without simultaneous feeding of glucose or amino acids. Methods: Mesenteric lymph duct was cannulated in male SD rats and a feeding tube was inserted into the duodenum through the fundus of the stomach. Postoperatively, the rats were intraduodenally infused with saline. On the day after surgery, the saline infusion was replaced by a lipid infusion containing 40 μmol/h (35.4 mg/h) of triolein, 2.74 μmol/h (1.06 mg/h) of cholesterol, 7.8 μmol/h ( 6.08 mg/h) of egg lecithin with 300 mg/h of glucose (the Glucose group), with 150 mg/h of amino acids derived from medical amino acid injections (the Amino Acids group), or without any additive (the Control group). The lipid emulsion was infused at 3mL/h for 8 hours. Results: The amounts of triglyceride transported in lymph for 8 hours were 185 ± 12 (mean ± SE) mg in the Amino Acids group (n=4), 175 ± 3 mg in the Glucose group (n=5), and 147 ± 7 mg in the Control group (n=4), respectively, with a statistically significant difference (p &lt; 0.05) among the groups. The amount of cholesterol transported in lymph for 8 hours were 7.30 ± 0.63 mg in the Amino Acids group, 6.82 ± 0.46 mg in the Glucose group, and 5.96 ± 0.17 mg in the Control group, respectively. The amount of phosphatidylcholine transported in lymph for 8 hours were 16.4 ± 1.0 mg in the Amino Acids group, 15.7 ± 0.4 mg in the Glucose group, and 12.4 ± 0.3 mg in the Control group, respectively, with a statistically significant difference (p &lt; 0.01) among the groups. Conclusions: Simultaneous glucose or amino acids feeding enhanced lymphatic lipid transport in the rat intestine during lipid feeding.

Abstract 543: Enhancement of Rat Lymphatic Lipid Transport by Glucose Ingestion

The intestine is a major source of lipid in the body. The mechanisms of absorption, intracellular metabolism, and lymphatic transport of intestinally derived lipid have been investigated vigorously. The lipid in the intestinal lumen is supplied as bile and food, the latter of which usually contains many kinds of nutrients. Although the intestinal lipid absorption physiologically occurs with the absorption of other nutrients, little is known about the effect of simultaneously absorbed other nutrients on lipid absorption in the intestine. The aim of this study is to clarify the effect of glucose ingestion on lipid transport in rat mesenteric lymph. The glucose is derived from carbohydrate which is a major constituent of food. A mesenteric lymph fistula was made in male Sprague-Dawley rats and the duodenum was cannulated through the fundus of stomach. Postoperatively, the rats were allowed to recover in restraint cages and intraduodenally infused with saline. On the day after surgery, the saline infusion was replaced by a lipid infusion containing 35.4 mg/h of triolein, added with cholesterol, egg lecithin, and taurocholate, emulsified in phosphate-buffered saline with (the experimental group) or without (the control group) 10% glucose. While the lipid emulsion was infused for 8 hours, lymph was collected before and hourly during the lipid infusion. The concentrations of glucose, triglyceride (TG), cholesterol, and phosphatidylcholine (PC) in lymph were measured enzymatically. The glucose concentration in lymph increased in the experimental group. The lymphatic glucose concentrations in 8 h were 338 ± 79 (mean ± SE) mg/dl in the experimental group (n = 5) and 152 ± 14 mg/dl in the control group (n = 4), respectively. The amount of TG transported in lymph for 8 hours were 175 ± 3 mg in the experimental group and 147 ± 7 mg in the control group, respectively, with a statistically significant difference (p &lt; 0.01). PC was also transported in lymph significantly more in the experimental group than in the control group. In conclusion, simultaneous glucose feeding enhanced lymphatic lipid transport in the rat intestine.

Apolipoprotein A-V is present in bile and its secretion increases with lipid absorption in Sprague-Dawley rats.

Apolipoprotein (apo) A-V is a protein synthesized only in the liver that dramatically modulates plasma triglyceride levels. Recent studies suggest a novel role for hepatic apoA-V in regulating the absorption of dietary triglycerides, but its mode of action on the gut remains unknown. The aim of this study was to test for apoA-V in bile and to determine whether its secretion is regulated by dietary lipids. After an overnight recovery, adult male Sprague-Dawley bile fistula rats indeed secreted apoA-V into bile at a constant rate under fasting conditions. An intraduodenal bolus of intralipid (n = 12) increased the biliary secretion of apoA-V but not of other apolipoproteins, such as A-I, A-IV, B, and E. The lipid-induced increase of biliary apoA-V was abolished under conditions of poor lymphatic lipid transport, suggesting that the stimulation is regulated by the magnitude of lipids associated with chylomicrons transported into lymph. We also studied the secretion of apoA-V into bile immediately following bile duct cannulation. Biliary apoA-V increased over time (∼6-fold increase at hour 16, n = 8) but the secretions of other apolipoproteins remained constant. Replenishing luminal phosphatidylcholine and taurocholate (n = 9) only enhanced apoA-V secretion in bile, suggesting that the increase was not due to depletion of phospholipids or bile salts. This is the first study to demonstrate that apoA-V is secreted into bile, introducing a potential route of delivery of hepatic apoA-V to the gut lumen. Our study also reveals the uniqueness of apoA-V secretion into bile that is regulated by mechanisms different from other apolipoproteins.

Dietary egg white protein inhibits lymphatic lipid transport in thoracic lymph duct-cannulated rats.

Dietary egg white protein (EWP) decreases serum cholesterol levels. We previously showed that EWP decreased cholesterol absorption in the intestine. Rats subjected to permanent lymph duct cannulation were used to investigate the effects of dietary EWP on lipid transport. They were fed diets with 20% EWP and casein, and their lymph was collected to quantify lymphatic lipid levels. Dietary EWP decreased lymphatic cholesterol transport compared with casein. It was previously shown that EWP excluded cholesterol from bile acid micelles. Therefore, pepsin-hydrolyzed EWP and casein were prepared. EWP was not completely digested. Ovalbumin, which is the most abundant protein in EWP, showed resistance to digestion by pepsin. This study investigated the effects of EWP pepsin hydrolysate (EWP-ph) on cholesterol micellar solubility, cholesterol transfer from the micellar to the oil phase, water-holding capacity (WHC), settling volume in water (SV), and relative viscosity and compared them with the effects of casein pepsin hydrolysate (C-ph). EWP-ph significantly decreased the micellar solubility and transfer rate and increased the WHC, SV, and relative viscosity compared with C-ph. Moreover, the pepsin hydrolysate of ovalbumin, a major protein in EWP, played a role in decreasing cholesterol micellar solubility, leading to the inhibition of cholesterol absorption. In conclusion, dietary EWP decreased cholesterol intestinal absorption by exerting combined effects of these physicochemical properties in the gut.

Overexpression of apolipoprotein C-III decreases secretion of dietary triglyceride into lymph.

Apolipoprotein C‐III (apoC‐III) is not only predominantly synthesized by the liver but also by the small intestine. Because apoC‐III is secreted from the intestine on the chylomicron along with lipid absorption, we questioned whether apoC‐III might play a role in intestinal lipid absorption and/or transport. Using both wild‐type (WT) and apoC‐III transgenic (apoC‐III Tg) mice, we showed that apoC‐III Tg mice have decreased lymphatic lipid transport compared with WT mice in response to an intraduodenal infusion of radiolabeled lipid. This is associated with accumulation of radiolabeled lipids in the luminal compartment of the apoC‐III Tg mice, indicating delayed lipid uptake from the lumen. The total amount of radioactive lipids in the mucosal compartment did not differ between apoC‐III Tg and WT mice, but the lipid distribution analysis indicated a predominance of free fatty acids and monoacylglycerol in the mucosa of apoC‐III Tg mice, implying impaired esterification capacity. Thus, the mechanisms underlying the reduced lymphatic lipid transport in apoC‐III Tg mice involve both a delayed lipid uptake into enterocytes, as well as impaired esterification to form triglyceride in the mucosa. These data document a novel role for apoC‐III in the uptake, re‐esterification, and lymphatic transport of dietary lipids in the intestine.

Lymphatic Transport of α‐Linolenic Acid and Its Conversion to Long Chain n‐3 Fatty Acids in Rats Fed Microemulsions of Linseed Oil

In the present study we evaluated the uptake of ALA and its conversion to EPA+DHA in rats given linseed oil (LSO) in native form or as a microemulsion in whey protein or in lipoid. In a single oral dose study in which rats maintained on rodent pellets deficient in ω-3 fatty acids were intubated with 0.35g LSO in lipoid, the amount of ALA present in lymph was increased reaching a maximum concentration of 16.23mg/ml at 2.5h. The amount of ALA present in lymph was increased to a maximum level of 10.95mg/ml at 4h in rats given LSO as a microemulsion in whey protein. When LSO was given without emulsification, the amount of ALA present in lymph was found to reach a maximum level of 7.08mg/ml at 6h. A similar result was observed when weaning rats were intubated with 0.15g of LSO per day for a period of 60days. Higher levels of ALA by 41 and 103% were observed in lymph lipids of rats given microemulsions of LSO in whey protein and in lipoid respectively as compared to rats given LSO without pre-emulsification. Very little conversion of ALA to EPA and DHA was observed in lymph lipids but higher amounts of EPA+DHA was observed in liver and serum of rats given LSO in microemulsion form. This study indicated that ALA concentration in lymph lipids was increased when LSO was given in microemulsion form in lipoid and further conversion to EPA and DHA was facilitated in liver and serum.