Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption.     

Intestinal alkaline phosphatase: selective endocytosis from the enterocyte brush border during fat absorption

Absorption of dietary fat in the small intestine is accompanied by a rise of intestinal alkaline phosphatase (IAP) in the serum and of secretion of IAP-containing surfactant-like particles from the enterocytes. In the present work, fat absorption was studied in organ cultured mouse intestinal explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via clathrin-coated pits. By 60 min, IAP was seen in subapical endosomes and along membranes surrounding fat droplets. IAP is a well-known lipid raft-associated protein, and fat absorption was accompanied by a marked change in the density and morphology of the detergent-resistant membranes harboring IAP. A lipid analysis revealed that fat absorption caused a marked increase in the microvillar membrane contents of free fatty acids. In conclusion, fat absorption rapidly induces a transient clathrin-dependent endocytosis via coated pits from the enterocyte brush border. The process selectively internalizes IAP and may contribute to the appearance of the enzyme in serum and surfactant-like particles.     

An Updated Perspective on the Dual-Track Model of Enterocyte Fat Metabolism

The small intestinal epithelium has classically been envisioned as a conduit for nutrient absorption, but appreciation is growing for a larger and more dynamic role for enterocytes in lipid metabolism. Considerable gaps remain in our knowledge of this physiology, but it appears that the enterocyte’s structural polarization dictates its behavior in fat partitioning, treating fat differently based on its absorption across the apical versus the basolateral membrane. In this review, we synthesize existing data and thought on this dual-track model of enterocyte fat metabolism through the lens of human integrative physiology. The apical track includes the canonical pathway of dietary lipid absorption across the apical brush-border membrane, leading to packaging and secretion of those lipids as chylomicrons. However, this track also reserves a portion of dietary lipid within cytoplasmic lipid droplets for later uses, including the “second-meal effect,” which remains poorly understood. At the same time, the enterocyte takes up circulating fats across the basolateral membrane by mechanisms that may include receptor-mediated import of triglyceride-rich lipoproteins or their remnants, local hydrolysis and internalization of free fatty acids, or enterocyte de novo lipogenesis using basolaterally absorbed substrates. The ultimate destinations of basolateral-track fat may include fatty acid oxidation, structural lipid synthesis, storage in cytoplasmic lipid droplets, or ultimate resecretion, although the regulation and purposes of this basolateral track remain mysterious. We propose that the enterocyte integrates lipid flux along both of these tracks in order to calibrate its overall program of lipid metabolism.     

DGAT1 deficiency disrupts lysosome function in enterocytes during dietary fat absorption

Enterocytes, the absorptive cells of the small intestine, mediate the process of dietary fat absorption by secreting triacylglycerol (TAG) into circulation. When levels of dietary fat are high, TAG is stored in cytoplasmic lipid droplets (CLDs) and sequentially hydrolyzed for ultimate secretion. Mice with deficiency in acyl CoA: diacylglycerol acyltransferase 1 (Dgat1^−/− mice) were previously reported to have a reduced rate of intestinal TAG secretion and abnormal TAG accumulation in enterocyte CLDs. This unique intestinal phenotype is critical to their resistance to diet-induced obesity; however, the underlying mechanism remains unclear. Emerging evidence shows that lysosomal TAG hydrolysis contributes to autophagy-mediated CLD mobilization termed lipophagy, and when disrupted results in CLD accumulation. In order to study how lipophagy contributes to the unique intestinal phenotype of Dgat1^−/− mice, enterocytes from wild-type (WT) and Dgat1^−/− mice were examined at 2 and 6 h after oral oil gavage. Through ultrastructural analysis we observed TAG present within autophagic vesicles (AVs) in mouse enterocytes, suggesting the role of lipophagy in intestinal CLD mobilization during dietary fat absorption. Furthermore, we found that Dgat1^−/− mice had abnormal TAG accumulation within AVs and less acidic lysosomes compared to WT mice. Together these findings suggest that the delayed dietary fat absorption seen in Dgat1^−/− mice is, in part, due to the dysregulated flux of autophagy-mediated CLD mobilization and impairment of lysosomal acidification in enterocytes. The present study highlights the critical role of lysosome in enterocyte CLD mobilization for proper dietary fat absorption.     

Differential association of adipophilin and TIP47 proteins with cytoplasmic lipid droplets in mouse enterocytes during dietary fat absorption

Recently, we found that enterocytes dynamically store triglycerides (TGs) in cytoplasmic lipid droplets (CLDs) during dietary fat absorption. A dynamic pool of TG in the form of CLDs which expands and depletes relative to time post dietary fat challenge is present in the absorptive cells of the small intestine, enterocytes. To identify cellular factors which may play a role in the regulation of this dynamic process we investigated the expression and localization of a lipid droplet associated protein family, PAT proteins, in enterocytes of mice chronically and acutely challenged by dietary fat. We found that adipophilin and Tip47 are the only PAT genes present in mouse intestinal mucosa and both genes are present at higher levels after high-fat challenges. We found TIP47 protein present in the intestine from chow and high-fat challenged mice; however, adipophilin protein was only present after high-fat challenges. In addition, TIP47 protein level was higher after an acute than a chronic high-fat challenge whereas adipophilin protein level was higher after a chronic than an acute high-fat challenge. We co-imaged TG in CLDs using CARS microscopy and TIP47 or adipophilin using immunocytochemistry in isolated enterocytes from mice challenged chronically and acutely by high levels of dietary fat. TIP47, but not adipophilin, coats CLDs in enterocytes after an acute high-fat challenge suggesting that TIP47 plays a role in the synthesis of CLDs from newly synthesized TG at the beginning of the process of dietary fat absorption in enterocytes. Adipophilin, on the other hand, coats CLDs only in enterocytes of chronic high-fat fed mice suggesting that adipophilin may play a role in the stabilization of TG stored in CLDs in longer term. These results suggest distinct roles for TIP47 and adipophilin in dietary fat absorption.     

Dgat1 and Dgat2 regulate enterocyte triacylglycerol distribution and alter proteins associated with cytoplasmic lipid droplets in response to dietary fat

Enterocytes, the absorptive cells of the small intestine, mediate efficient absorption of dietary fat (triacylglycerol, TAG). The digestive products of dietary fat are taken up by enterocytes, re-esterified into TAG, and packaged on chylomicrons (CMs) for secretion into blood or temporarily stored within cytoplasmic lipid droplets (CLDs). Altered enterocyte TAG distribution impacts susceptibility to high fat diet associated diseases, but molecular mechanisms directing TAG toward these fates are unclear. Two enzymes, acyl CoA: diacylglycerol acyltransferase 1 (Dgat1) and Dgat2, catalyze the final, committed step of TAG synthesis within enterocytes. Mice with intestine-specific overexpression of Dgat1 (Dgat1^Int) or Dgat2 (Dgat2^Int), or lack of Dgat1 (Dgat1^−/−), were previously found to have altered intestinal TAG secretion and storage. We hypothesized that varying intestinal Dgat1 and Dgat2 levels alters TAG distribution in subcellular pools for CM synthesis as well as the morphology and proteome of CLDs. To test this we used ultrastructural and proteomic methods to investigate intracellular TAG distribution and CLD-associated proteins in enterocytes from Dgat1^Int, Dgat2^Int, and Dgat1^−/− mice 2 h after a 200 μl oral olive oil gavage. We found that varying levels of intestinal Dgat1 and Dgat2 altered TAG pools involved in CM assembly and secretion, the number or size of CLDs present in enterocytes, and the enterocyte CLD proteome. Overall, these results support a model where Dgat1 and Dgat2 function coordinately to regulate the process of dietary fat absorption by preferentially synthesizing TAG for incorporation into distinct subcellular TAG pools in enterocytes.     

A dynamic, cytoplasmic triacylglycerol pool in enterocytes revealed by ex vivo and in vivo coherent anti-Stokes Raman scattering imaging

The absorptive cells of the small intestine, enterocytes, are not generally thought of as a cell type that stores triacylglycerols (TGs) in cytoplasmic lipid droplets (LDs). We revisit TG metabolism in enterocytes by ex vivo and in vivo coherent anti-Stokes Raman scattering (CARS) imaging of small intestine of mice during dietary fat absorption (DFA). We directly visualized the presence of LDs in enterocytes. We determined lipid amount and quantified LD number and size as a function of intestinal location and time post-lipid challenge via gavage feeding. The LDs were confirmed to be primarily TG by biochemical analysis. Combined CARS and fluorescence imaging indicated that the large LDs were located in the cytoplasm, associated with the tail-interacting protein of 47 kDa. Furthermore, in vivo CARS imaging showed real-time variation in the amount of TG stored in LDs through the process of DFA. Our results highlight a dynamic, cytoplasmic TG pool in enterocytes that may play previously unexpected roles in processes, such as regulating postprandial blood TG concentrations.     

Recent advances in cytoplasmic lipid droplet metabolism in intestinal enterocyte

Processing of dietary fats in the intestine is a highly regulated process that influences whole-body energy homeostasis and multiple physiological functions. Dysregulated lipid handling in the intestine leads to dyslipidemia and atherosclerotic cardiovascular disease. In intestinal enterocytes, lipids are incorporated into lipoproteins and cytoplasmic lipid droplets (CLDs). Lipoprotein synthesis and CLD metabolism are inter-connected pathways with multiple points of regulation. This review aims to highlight recent advances in the regulatory mechanisms of lipid processing in the enterocyte, with particular focus on CLDs. In-depth understanding of the regulation of lipid metabolism in the enterocyte may help identify therapeutic targets for the treatment and prevention of metabolic disorders.     

Lipidomic and Spatio-Temporal Imaging of Fat by Mass Spectrometry in Mice Duodenum during Lipid Digestion

Intestinal absorption of dietary fat is a complex process mediated by enterocytes leading to lipid assembly and secretion of circulating lipoproteins as chylomicrons, vLDL and intestinal HDL (iHDL). Understanding lipid digestion is of importance knowing the correlation between excessive fat absorption and atherosclerosis. By using time-of-flight secondary ion mass spectrometry (TOF-SIMS), we illustrated a spatio-temporal localization of fat in mice duodenum, at different times of digestion after a lipid gavage, for the first time. Fatty acids progressively increased in enterocytes as well as taurocholic acid, secreted by bile and engaged in the entero-hepatic re-absorption cycle. Cytosolic lipid droplets (CLD) from enterocytes were originally purified separating chylomicron-like, intermediate droplets and smaller HDL-like. A lipidomic quantification revealed their contents in triglycerides, free and esterified cholesterol, phosphatidylcholine, sphingomyelin and ceramides but also in free fatty acids, mono- and di-acylglycerols. An acyl-transferase activity was identified and the enzyme monoacylglycerol acyl transferase 2 (MGAT2) was immunodetected in all CLD. The largest droplets was also shown to contain the microsomal triglyceride transfer protein (MTTP), the acyl-coenzyme A-cholesterol acyltransferases (ACAT) 1 and 2, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). This highlights the fact that during the digestion of fats, enterocyte CLD contain some enzymes involved in the different stages of the metabolism of diet fatty acids and cholesterol, in anticipation of the crucial work of endoplasmic reticulum in the process. The data further underlines the dual role of chylomicrons and iHDL in fat digestion which should help to efficiently complement lipid-lowering therapy.     

MGAT2, a monoacylglycerol acyltransferase expressed in the small intestine

Acyl CoA:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, a precursor of triacylglycerol. In the intestine, MGAT plays a major role in the absorption of dietary fat by catalyzing the resynthesis of triacylglycerol in enterocytes. This resynthesis is required for the assembly of lipoproteins that transport absorbed fat to other tissues. Despite intense efforts, a gene encoding an intestinal MGAT has not been found. Previously, we identified a gene encoding MGAT1, which in mice is expressed in the stomach, kidney, adipose tissue, and liver but not in the intestine. We now report the identification of homologous genes in humans and mice encoding MGAT2. Expression of the MGAT2 cDNA in either insect or mammalian cells markedly increased MGAT activity in cell membranes. MGAT activity was proportional to the level of MGAT2 protein expressed, and the amount of diacylglycerol produced depended on the concentration of MGAT substrates (fatty acyl CoA or monoacylglycerol). In humans, the MGAT2 gene is highly expressed in the small intestine, liver, stomach, kidney, colon, and white adipose tissue; in mice, it is expressed predominantly in the small intestine. The discovery of the MGAT2 gene will facilitate studies to determine the functional role of MGAT2 in fat absorption in the intestine and to determine whether blocking MGAT activity in enterocytes is a feasible approach to inhibit fat absorption and treat obesity.     

Postprandial morphological response of the intestinal epithelium of the Burmese python (Python molurus)

The postprandial morphological changes of the intestinal epithelium of Burmese pythons were examined using fasting pythons and at eight time points after feeding. In fasting pythons, tightly packed enterocytes possess very short microvilli and are arranged in a pseudostratified fashion. Enterocyte width increases by 23% within 24 h postfeeding, inducing significant increases in villus length and intestinal mass. By 6 days postfeeding, enterocyte volume had peaked, following as much as an 80% increase. Contributing to enterocyte hypertrophy is the cellular accumulation of lipid droplets at the tips and edges of the villi of the proximal and middle small intestine, but which were absent in the distal small intestine. At 3 days postfeeding, conventional and environmental scanning electron microscopy revealed cracks and lipid extrusion along the narrow edges of the villi and at the villus tips. Transmission electron microscopy demonstrated the rapid postprandial lengthening of enterocyte microvilli, increasing 4.8-fold in length within 24 h, and the maintaining of that length through digestion. Beginning at 24 h postfeeding, spherical particles were found embedded apically within enterocytes of the proximal and middle small intestine. These particles possessed an annular-like construction and were stained with the calcium-stain Alizarine red S suggesting that they were bone in origin. Following the completion of digestion, many of the postprandial responses were reversed, as observed by the atrophy of enterocytes, the shortening of villi, and the retraction of the microvilli. Further exploration of the python intestine will reveal the underlying mechanisms of these trophic responses and the origin and fate of the engulfed particles.     

Increased dietary triacylglycerol markedly enhances the ability of isolated rabbit enterocytes to secrete chylomicrons: an effect related to dietary fatty acid composition.

Dietary fats are efficiently absorbed in the small intestine and transported into the blood via the lymph as chylomicrons, despite enormous variations in the amount and composition of the dietary lipid. The aim of the present study was to investigate how enterocytes respond to increased dietary fats of different composition. Rabbits were fed a low fat chow diet, and chow supplemented with sunflower oil (high n-6 polyunsaturated fatty acids), fish oil (high n-3 polyunsaturated fatty acids), or an oil mixture of a composition similar to that of the typical western diet. Feeding fat for 2 weeks markedly stimulated the ability of the isolated enterocytes to synthesize and secrete apolipoprotein B48, triacylglycerol, and cholesteryl ester (up to 18-, 50-, and 80-fold, respectively) in particles of chylomicron density. The magnitude of stimulation was sunflower oil > western diet lipid > fish oil. Single doses of lipid given 18 h prior to isolation of enterocytes stimulated chylomicron secretion by only 10% of that observed after 2 weeks of dietary supplementation. Enterocytes are replaced rapidly (half-life 1-2 days) by cells which move from the crypts to the tips of the villi, where absorption of nutrients takes place.Our observations suggest that dietary lipids modulate the function of enterocytes as they move from the crypts, so that the cells are 'turned-on' to lipid absorption. The results also show that diets of different fatty acid composition vary in their effects.     

Similar bioavailability and lymphatic transport of benzo(a)pyrene when administered to rats in different amounts of dietary fat

This study assessed the effect of concomitant lipid absorption on the bioavailability and lymphatic transport of benzo(a)pyrene (BP), a carcinogenic polycyclic aromatic hydrocarbon (PAH). Conscious, male Sprague-Dawley rats, equipped with biliary and mesenteric lymphatic catheters received intraduodenally a dose of 0.4 mumoles 3H-labeled BP completely dissolved in either 50 mumoles or 500 mumoles of olive oil. Diversion of mesenteric lymph allowed biliary and urinary excretion of 3H to be used as an indirect measurement of relative 3H portal transport. Total radiolabel recovered in a 24-hr period in each group was 20.0 +/- 2.6% of the 3H dose given in 50 mumoles of oil, and 17.0 +/- 1.0% of the 3H dose administered in 500 mumoles of oil. In animals receiving the low-fat test meal, 79.4 +/- 1.4% of the recovered radiolabel was found in bile; the corresponding value for the high fat dose was 78.5 +/- 2.6%. Thus a tenfold variation in the mass of the carrier vehicle (triglyceride oil) did not significantly effect the disposition of BP, and portal, not lymphatic transport, was the major route of post-absorptive transport. Although the chylomicrons produced from both fat doses were initially contaminated with BP, within 1-1.5 hr the radioactivity in lymph began to drop such that by 3 hr in the animals fed high fat, the chylomicrons were essentially free of BP. These results show that the rat enterocyte quickly adapts to PAH-contaminated dietary fat, even during the assimilation of a single dose of fat. Presumably, during the post-absorptive synthesis of chylomicrons from pre-chylomicrons, BP is metabolized and removed from the triglyceride oil droplets.     

Dietary fat sensing via fatty acid oxidation in enterocytes: possible role in the control of eating

Various mechanisms detect the presence of dietary triacylglycerols (TAG) in the digestive tract and link TAG ingestion to the regulation of energy homeostasis. We here propose a novel sensing mechanism with the potential to encode dietary TAG-derived energy by translating enterocyte fatty acid oxidation (FAO) into vagal afferent signals controlling eating. Peripheral FAO has long been implicated in the control of eating (141). The prevailing view was that mercaptoacetate (MA) and other FAO inhibitors stimulate eating by modulating vagal afferent signaling from the liver. This concept has been challenged because hepatic parenchymal vagal afferent innervation is scarce and because experimentally induced changes in hepatic FAO often fail to affect eating. Nevertheless, intraperitoneally administered MA acts in the abdomen to stimulate eating because this effect was blocked by subdiaphragmatic vagal deafferentation (21), a surgical technique that eliminates all vagal afferents from the upper gut. These and other data support a role of the small intestine rather than the liver as a FAO sensor that can influence eating. After intrajejunal infusions, MA also stimulated eating in rats through vagal afferent signaling, and after infusion into the superior mesenteric artery, MA increased the activity of celiac vagal afferent fibers originating in the proximal small intestine. Also, pharmacological interference with TAG synthesis targeting the small intestine induced a metabolic profile indicative of increased FAO and inhibited eating in rats on a high-fat diet but not on chow. Finally, cell culture studies indicate that enterocytes oxidize fatty acids, which can be modified pharmacologically. Thus enterocytes may sense dietary TAG-derived fatty acids via FAO and influence eating through changes in intestinal vagal afferent activity. Further studies are necessary to identify the link between enterocyte FAO and vagal afferents and to examine the specificity and potential physiological relevance of such a mechanism.     

Fat intestinal absorption in the catfish--a histochemical study in glycol methacrylate embedded tissue

The intestinal absorption of lipids was investigated in plastic sections from glycol methacrylate embedded intestine after fat administration. In the catfish, the lipids are absorbed by the enterocytes of the proximal intestinal segment, thus forming fat cytoplasmic inclusions that were demonstrated by Sudan black B staining. The histochemical characterization of lipids by the Nile blue sulphate test revealed the neutral or triglyceride nature of the cytoplasmic droplets, both after the corn oil and oleic acid feeding. There is lipid accumulation in the lamina propria and lymphatic vessels.     

Fat absorptive processes in the intestine of the Antarctic fish Notothenia coriiceps (Richardson, 1844)

Absorption of lipids by the enterocytes of Notothenia coriiceps, an omnivorous Antarctic and subAntarctic fish, was studied by light and electron microscopy. The lipids are absorbed by the anterior and middle intestine segments. They appear as fat droplets that measure from 0.5 to 7 μm of diameter and which accumulate in the apical cytoplasm within the first 24 h and seem to be the main fat storage form in the enterocytes. Fat is also observed as lipid particles with 60–300 nm inside the rough endoplasmic reticulum and cytoplasmic smooth vesicles. The epithelial intercellular space and the mucosal inner lamina contain lipid particles, which probably are the fat transport form. Our observations show that an intense lipid absorptive process takes place in N. coriiceps digestive system, due to the great extension of the intestine involved and due to the great lipid accumulation found in the epithelial compartment.     

DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis

Dietary triacylglycerols are a major source of energy for animals. The absorption of dietary triacylglycerols involves their hydrolysis to free fatty acids and monoacylglycerols in the intestinal lumen, the uptake of these products into enterocytes, the resynthesis of triacylgylcerols, and the incorporation of newly synthesized triacylglycerols into nascent chylomicrons for secretion. In enterocytes, the final step in triacylglycerol synthesis is believed to be catalyzed primarily through the actions of acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. In this study, we analyzed intestinal triacylglycerol absorption and chylomicron synthesis and secretion in DGAT1-deficient (Dgat1(-/-)) mice. Surprisingly, DGAT1 was not essential for quantitative dietary triacylglycerol absorption, even in mice fed a high fat diet, or for the synthesis of chylomicrons. However, Dgat1(-/-) mice had reduced postabsorptive chylomicronemia (1 h after a high fat challenge) and accumulated neutral-lipid droplets in the cytoplasm of enterocytes when chronically fed a high fat diet. These results suggest a reduced rate of triacylglycerol absorption in Dgat1(-/-) mice. Analysis of intestine from Dgat1(-/-) mice revealed activity for two other enzymes, DGAT2 and diacylglycerol transacylase, that catalyze triacylglycerol synthesis and apparently help to compensate for the absence of DGAT1. Our findings indicate that multiple mechanisms for triacylglycerol synthesis in the intestine facilitate triacylglycerol absorption.