Intestinal Lipid Handling Research Articles

GLP-2 regulation of intestinal lipid handling.

Lipid handling in the intestine is important for maintaining energy homeostasis and overall health. Mishandling of lipids in the intestine contributes to dyslipidemia and atherosclerotic cardiovascular diseases. Despite advances in this field over the past few decades, significant gaps remain. The gut hormone glucagon-like peptide-2 (GLP-2) has been shown to play pleotropic roles in the regulation of lipid handling in the intestine. Of note, GLP-2 exhibits unique actions on post-prandial lipid absorption and post-absorptive release of intestinally stored lipids. This review aims to summarize current knowledge in how GLP-2 regulates lipid processing in the intestine. Elucidating the mechanisms of GLP-2 regulation of intestinal lipid handling not only improves our understanding of GLP-2 biology, but also provides insights into how lipids are processed in the intestine, which offers opportunities for developing novel strategies towards prevention and treatment of dyslipidemia and atherosclerotic cardiovascular diseases.

Adaptation to short-term extreme fat consumption alters intestinal lipid handling in male and female mice

The small intestine is a highly adaptable organ serving as both a barrier to the external environment and a conduit for nutrient absorption. Enterocytes package dietary triglycerides (TG) into chylomicrons for transport into circulation; the remaining TGs are stored in cytosolic lipid droplets (CLDs). The current study aimed to characterize the impact of diet composition on intestinal lipid handling in male and female wild-type mice. Mice were continued on their grain-based diet (GBD) and switched to either a high-fat, high cholesterol Western-style diet (WD) or a ketogenic diet (KD) for 3 or 5 weeks. KD-fed mice displayed significantly higher plasma TG levels in response to an olive oil gavage than WD- and GBD-fed mice; TG levels were ~2-fold higher in male KD-fed mice than female KD-fed mice. Poloxamer-407 experiments revealed enhanced intestinal-TG secretion rates in male mice fed a KD upon olive oil gavage, whereas secretion rates were unchanged in female mice. Surprisingly, jejunal CLD size and TG mass after oil gavage were similar among the groups. At fasting, TG mass was significantly higher in the jejunum of male KD-fed mice and the duodenum of female KD-fed mice, providing increased substrate for chylomicron formation. In addition to greater fasting intestinal TG stores, KD-fed male mice displayed longer small intestinal lengths, while female mice displayed markedly longer jejunal villi lengths. After 5 weeks of diet, 12 h fasting-2 h refeeding experiments revealed jejunal TG levels were similar between diet groups in male mice; however, in female mice, jejunal TG mass was significantly higher in KD-fed mice compared to GBD- and WD-fed mice. These experiments reveal that KD feeding promotes distinct morphological and functional changes to the murine small intestine compared to the WD diet. Moreover, changes to intestinal lipid handling in response to carbohydrate and protein restriction manifest differently in male and female mice.

High-throughput LC-MS method to investigate postprandial lipemia: considerations for future precision nutrition research.

Elevated postprandial lipemia is an independent risk factor for cardiovascular disease, yet methods to quantitate postmeal handling of dietary lipids in humans are limited. This study tested a new method to track dietary lipid appearance using a stable isotope tracer (2H11-oleate) in liquid meals containing three levels of fat [low fat (LF), 15 g; moderate fat (MF), 30 g; high fat (HF), 60 g]. Meals were fed to 12 healthy men [means ± SD, age 31.3 ± 9.2 yr, body mass index (BMI) 24.5 ± 1.9 kg/m2] during four randomized study visits; the HF meal was administered twice for reproducibility. Blood was collected over 8 h postprandially, triglyceride (TG)-rich lipoproteins (TRL), and particles with a Svedberg flotation rate >400 (Sf > 400, n = 8) were isolated by ultracentrifugation, and labeling of two TG species (54:3 and 52:2) was quantified by LC-MS. Total plasma TRL-TG concentrations were threefold greater than Sf > 400-TG. Both Sf > 400- and TRL-TG 54:3 were present at higher concentrations than 52:2, and singly labeled TG concentrations were higher than doubly labeled. Furthermore, TG 54:3 and the singly labeled molecules demonstrated higher plasma absolute entry rates differing significantly across fat levels within a single TG species (P < 0.01). Calculation of fractional entry showed no significant differences in label handling supporting the utility of either TG species for appearance rate calculations. These data demonstrate the utility of labeling research meals with stable isotopes to investigate human postprandial lipemia while simultaneously highlighting the importance of examining individual responses. Meal type and timing, control of prestudy activities, and effects of sex on outcomes should match the research goals. The method, optimized here, will be beneficial to conduct basic science research in precision nutrition and clinical drug development.NEW & NOTEWORTHY A novel method to test human intestinal lipid handling using stable isotope labeling is presented and, for the first time, plasma appearance and lipid turnover were quantified in 12 healthy men following meals with varying amounts of fat. The method can be applied to studies in precision nutrition characterizing individual response to support basic science research or drug development. This report discusses key questions for consideration in precision nutrition that were highlighted by the data.

Role of the Gut in Diabetic Dyslipidemia.

Type 2 diabetes (T2D) is associated with increased risk of cardiovascular disease (CVD). In insulin resistant states such as the metabolic syndrome, overproduction and impaired clearance of liver-derived very-low-density lipoproteins and gut-derived chylomicrons (CMs) contribute to hypertriglyceridemia and elevated atherogenic remnant lipoproteins. Although ingested fat is the major stimulus of CM secretion, intestinal lipid handling and ultimately CM secretory rate is determined by numerous additional regulatory inputs including nutrients, hormones and neural signals that fine tune CM secretion during fasted and fed states. Insulin resistance and T2D represent perturbed metabolic states in which intestinal sensitivity to key regulatory hormones such as insulin, leptin and glucagon-like peptide-1 (GLP-1) may be altered, contributing to increased CM secretion. In this review, we describe the evidence from human and animal models demonstrating increased CM secretion in insulin resistance and T2D and discuss the molecular mechanisms underlying these effects. Several novel compounds are in various stages of preclinical and clinical investigation to modulate intestinal CM synthesis and secretion. Their efficacy, safety and therapeutic utility are discussed. Similarly, the effects of currently approved lipid modulating therapies such as statins, ezetimibe, fibrates, and PCSK9 inhibitors on intestinal CM production are discussed. The intricacies of intestinal CM production are an active area of research that may yield novel therapies to prevent atherosclerotic CVD in insulin resistance and T2D.

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.

Propionate promotes intestinal lipolysis and metabolic benefits via AMPK/LSD1 pathway in mice.

Dietary fibers and their microbial fermentation products short chain fatty acids promote metabolic benefits, but the underlying mechanisms are still unclear. Recent studies indicate that intestinal lipid handling is under regulatory control and has broad influence on whole body energy homeostasis. Here we reported that dietary inulin and propionate significantly decreased whole body fat mass without affecting food intake in mice fed with chow diet. Meanwhile, triglyceride (TG) content was decreased and lipolysis genes expressions, such as adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and lysosomal acid lipase (LAL) were elevated in the jejunum and ileum of inulin and propionate treated mice. In vitro studies on Caco-2 cells showed propionate directly induced enterocyte ATGL, HSL and LAL gene expressions and decreased TG content, via activation of phosphorylation of AMP-activated protein kinase (p-AMPK) and lysine specific demethylase 1 (LSD1). Moreover, inulin and propionate could increase intestinal lipolysis under high fat diet (HFD) fed condition which contributed to the prevention of HFD-induced obesity. Our study suggests dietary fiber inulin and its microbial fermentation product propionate can regulate metabolic homeostasis through regulating intestinal lipid handling, which could provide a novel therapeutic target for both prevention and treatment of obesity.

Developments in intestinal cholesterol transport and triglyceride absorption.

To discuss recent advances in research focused on intestinal lipid handling. An important strategy in reducing atherosclerosis and risk of cardiovascular events is to increase the rate of reverse cholesterol transport, including its final step; cholesterol excretion from the body. The rate of removal is determined by a complex interplay between the factors involved in regulation of intestinal cholesterol absorption. One of these factors is a process known as transintestinal cholesterol excretion. This pathway comprises transport of cholesterol directly from the blood, through the enterocyte, into the intestinal lumen. In humans, this pathway accounts for 35% of cholesterol excretion in the feces. Mechanistic studies in mice revealed that, activation of the bile acid receptor farnesoid X receptor increases cholesterol removal via the transintestinal cholesterol excretion pathway as well as decreases plasma cholesterol and triglyceride providing an interesting target for treatment of dyslipidemia in humans. The physical chemical properties of bile acids are under control of farnesoid X receptor and determine intestinal cholesterol and triglyceride solubilization as well as absorption, providing a direct link between these two important factors in the pathogenesis of cardiovascular disease. Besides bile acids, intestinal phospholipids are important for luminal lipid solubilization. Interestingly, phospholipid remodeling through LPCAT3 was shown to be pivotal for uptake of fatty acids by enterocytes, which may provide a mechanistic handle for therapeutic intervention. The importance of the intestine in control of cholesterol and triglyceride homeostasis is increasingly recognized. Recently, novel factors involved in regulation of cholesterol excretion and intestinal triglyceride and fatty acid uptake have been reported and are discussed in this short review.

Role of Intestinal LXRα in Regulating Post-prandial Lipid Excursion and Diet-Induced Hypercholesterolemia and Hepatic Lipid Accumulation.

Post-prandial hyperlipidemia has emerged as a cardiovascular risk factor with limited therapeutic options. The Liver X receptors (Lxrs) are nuclear hormone receptors that regulate cholesterol elimination. Knowledge of their role in regulating the absorption and handling of dietary fats is incomplete. The purpose of this study was to determine the role of intestinal Lxrα in post-prandial intestinal lipid transport. Using Lxrα knockout (nr1h3−/−) and intestine-limited Lxrα over-expressing [Tg(fabp2a:EGFP-nr1h3)] zebrafish strains, we measured post-prandial lipid excursion with live imaging in larvae and physiological methods in adults. We also conducted a long-term high-cholesterol dietary challenge in adults to examine the chronic effect of modulating nr1h3 gene dose on the development of hypercholesterolemia and hepatic lipid accumulation. Over-expression of Lxrα in the intestine delays the transport of ingested lipids in larvae, while deletion of Lxrα increases the rate of lipid transport. Pre-treating wildtype larvae with the liver-sparing Lxr agonist hyodeoxycholic acid also delayed the rate of intestinal lipid transport in larvae. In adult males, deletion of Lxrα accelerates intestinal transport of ingested lipids. Adult females showed higher plasma Lipoprotein lipase (Lpl) activity compared to males, and lower post-gavage blood triacylglycerol (TAG) excursion. Despite the sexually dimorphic effect on acute intestinal lipid handling, Tg(fabp2a:EGFP-nr1h3) adults of both sexes are protected from high cholesterol diet (HCD)-induced hepatic lipid accumulation, while nr1h3−/− mutants are sensitive to the effects of HCD challenge. These data indicate that intestinal Lxr activity dampens the pace of intestinal lipid transport cell-autonomously. Selective activation of intestinal Lxrα holds therapeutic promise.

Abstract 206: Intestinal LXR Directs Absorbed Lipids to Storage

Post-prandial hypertriglyceridemia has emerged as a cardiovascular risk factor with limited therapeutic options. We have taken genetic and physiological approaches to identify and characertize novel factors that regulate intestinal lipid handling in the hope of revealing cellular pathways that dampen delivery of dietary lipids, opening the therapeutic window to blunting post-prandial lipid excursions. Liver X receptors (Lxrs) are master regulators of cholesterol catabolism, driving the elimination of cholesterol from the periphery to the lumen of the intestine. These nuclear hormone receptors are activated by oxysterol, ligands that accumulate when excess cholesterol is present. Loss of nr1h3 function causes anticipated gene regulatory changes and cholesterol intolerance, collectively reflecting high evolutionary conservation of zebrafish Lxra function. Intestinal nr1h3 activation delays transport of absorbed neutral lipids, with accumulation of neutral lipids in enterocyte cytoplasmic droplets, as visualized with histological stains, live fluorescent lipid probes, and ultrastructural analyses. This delay in transport of ingested neutral lipids protects animals over-expressing nr1h3 in the intestine from hypercholesterolemia and hepatic steatosis induced by a high-fat diet. On a gene regulatory level, Lxra induces expression of acsl3a , which encodes acyl-CoA synthetase long-chain family member 3a, a lipid droplet-anchored protein that directs fatty acyl chains into lipids. We have identified a gene regulatory network in enterocytes whereby the pace of delivery of dietary lipids is gated by Lxrs. Development of pharmacological agents to activate Lxrs has been hindered by synthetic Lxr agonists’ induction of hepatic lipogenesis and hypertriglyceridemia. The development of intestine-limited Lxr agonists could be of therapeutic value in blunting post-prandial hypertriglyceridemia.

Cyp8b1 ablation prevents Western diet-induced weight gain and hepatic steatosis because of impaired fat absorption.

Bile acids (BAs) are cholesterol derivatives that regulate lipid metabolism, through their dual abilities to promote lipid absorption and activate BA receptors. However, different BA species have varying abilities to perform these functions. Eliminating 12α-hydroxy BAs in mice via Cyp8b1 knockout causes low body weight and improved glucose tolerance. The goal of this study was to determine mechanisms of low body weight in Cyp8b1-/- mice. We challenged Cyp8b1-/- mice with a Western-type diet and assessed body weight and composition. We measured energy expenditure, fecal calories, and lipid absorption and performed lipidomic studies on feces and intestine. We investigated the requirement for dietary fat in the phenotype using a fat-free diet. Cyp8b1-/- mice were resistant to Western diet-induced body weight gain, hepatic steatosis, and insulin resistance. These changes were associated with increased fecal calories, due to malabsorption of hydrolyzed dietary triglycerides. This was reversed by treating the mice with taurocholic acid, the major 12α-hydroxylated BA species. The improvements in body weight and steatosis were normalized by feeding mice a fat-free diet. The effects of BA composition on intestinal lipid handling are important for whole body energy homeostasis. Thus modulating BA composition is a potential tool for obesity or diabetes therapy.

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.

Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function.

Modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein assembly by butyrate.

Short-chain fatty acids (SCFAs) are potent modulators of the growth, function, and differentiation of intestinal epithelia. In addition, high-fiber diets may protect against the development of atherosclerosis because of their cholesterol-lowering effects due, in large part, to SCFA production, liver sterol metabolism, and bile acid excretion. Although the small gut plays a major role in dietary fat transport and contributes substantially to plasma cholesterol and lipoprotein homeostasis, the impact of SCFAs on intestinal lipid handling remains unknown. In the present study, the modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein secretion by butyrate was investigated in Caco-2 cells plated on permeable polycarbonate filters, which permit separate access to the upper and lower compartments of the monolayers. Highly differentiated and polarized cells (20 days of culture) were incubated for 20 h with 20 mM butyrate in the apical medium. In the presence of [14C]oleic acid, butyrate led to a significant reduction of secreted, labeled triglycerides (27%; P < 0.01) and phospholipids (25%; P < 0.05). Similarly, butyrate significantly decreased the incorporation of [14C]acetate into exported cholesteryl ester (49%; P < 0.005). As expected from these results, with [14C]oleic acid as a precursor, butyrate significantly (P < 0.05) diminished the delivery of radiolabeled chylomicrons and very low-density lipoproteins. In parallel, [35S]methionine pulse labeling of Caco-2 cells revealed the concomitant inhibitory effect of butyrate on the synthesis of apolipoproteins B-48 (28%; P < 0.05) and A-I (32%; P < 0.01). Collectively, our data indicate that butyrate may influence lipid metabolism in Caco-2 cells, thus suggesting a potential regulation of intestinal fat absorption and circulating lipoprotein concentrations.

Tumor necrosis factor-alpha inhibits lipid and lipoprotein transport by Caco-2 cells

Cytokines, important mediators of inflammation, have been shown to cause disturbances in circulating and hepatic lipid metabolism. Although the intestine plays a major role in dietary fat transport and largely contributes to plasma lipoproteins, the effects of cytokines on intestinal lipid handling remain unknown. In the present study, the modulation of lipid, apoprotein, and lipoprotein synthesis and secretion by tumor necrosis factor-alpha (TNF-alpha) was investigated in Caco-2 cells. Highly differentiated and polarized cells (20 days in culture) were incubated for 20 h with recombinant human TNF-alpha (100-500 ng/ml). No cytotoxic effect of TNF-alpha cells was observed, as indicated by the determinations of Caco-2 cell viability and monolayer transepithelial resistance. Moreover, no differences in cell maturation (sucrase activity) or cell proliferation ([3H]thymidine incorporation and cell cycle analysis) were detected between treated and control cultures. Significant inhibition of lipid secretion by TNF-alpha was observed, with the greatest reduction at 500 ng/ml. TNF-alpha significantly decreased Caco-2 cell secretion of phospholipids (22%), triglycerides (30%), and cholesteryl ester (37%). It also significantly diminished the export of newly synthesized low-density lipoproteins (LDL; 20%) and high-density lipoproteins (HDL; 13%), with a lesser effect on very low-density lipoproteins (VLDL; 3%). The lipid composition of these lipoproteins was minimally affected. De novo synthesis of apo A-I, apo B-100, and apo B-48 was also markedly reduced by TNF-alpha. Sphingomyelinase activity was not increased and cell content of sphingomyelin was not altered, suggesting that inhibitory effects on lipid and apoprotein of TNF-alpha were not mediated by the ceramide pathway. Our results indicate that TNF-alpha may play a role in modulating intestinal lipid metabolism, thus affecting circulating lipoproteins.

The Effect of a New Calcium Channel Blocker (TA-3090) on Lipoprotein Profile and Intestinal Lipid Handling in Rodents

Recent interest has focused on findings that drugs used to lower blood pressure may adversely modify plasma lipids and lipoprotein metabolism. This observation may explain why pharmacologic control of hypertension has failed to reduce the incidence of morbidity and mortality from coronary artery disease. The present study aims to evaluate the effect of TA-3090, a new calcium channel blocker, on fasting plasma lipids and lipoproteins, as well as on processes of intestinal fat absorption. Rats were treated by gavage with TA-3090 (10 mg/kg twice daily) for 4 days and compared with controls (n = 6 per group). Plasma cholesterol was increased in the treated group to (mean +/- SE) 74 +/- 2 vs 60 +/- 4 mg/dl (P less than 0.01), due mainly to an increased high density lipoprotein-cholesterol level (50 +/- 2 vs 37 +/- 3 mg/dl, P less than 0.005). Notably plasma triglycerides (TG) and low density lipoprotein-cholesterol were not significantly affected. Another group of TA-3090-treated animals was given an intraduodenal fat meal, and the rise in plasma TG and chylomicrons followed over 4 hr. Postprandial hypertriglyceridemia and chylomicronemia were significantly lower at 2 hr (P less than 0.05) and 3 hr (P less than 0.01) compared with controls. In a separate group of animals, the addition of TA-3090 to a 2% intralipid infusion intraduodenally was associated with significantly reduced TG and chylomicron-TG transport into lymph (P less than 0.05). Furthermore, experiments in rats pretreated with TA-3090 intraperitoneally and then given 2% intralipid intraduodenally were shown to have a significant decrease in mean flow rate (27%), TG transport (31%) and chylomicron-TG output (37%), when compared with controls. In vitro studies using jejunal organ culture to examine the effect of TA-3090 on intracellular lipid synthesis and secretion revealed that the addition of the drug to the medium resulted in significantly decreased TG synthesis and secretion. These data suggest that TA-3090 could be effective in increasing HDL-cholesterol and reducing postprandial chylomicronemia. Our findings support a role for TA-3090 directly on enterocyte absorption and/or intracellular lipid transport, and thus indicate the importance of intracellular calcium on these processes.