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.     

Short-chain fatty acids suppress food intake by activating vagal afferent neurons

Fermentable carbohydrates including dietary fibers and resistant starch produce short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, through microbial fermentation in the intestine of rodents and humans. Consumption of fermentable carbohydrate and SCFAs suppress food intake, an effect involving the brain. However, their signaling pathway to the brain remains unclear. Vagal afferents serve to link intestinal information to the brain. In the present study, we explored possible role of vagal afferents in the anorexigenic effect of SCFAs. Intraperitoneal (ip) injection of three SCFA molecules (6 mmol/kg) suppressed food intake in fasted mice with the rank order of butyrate > propionate > acetate. The suppressions of feeding by butyrate, propionate and acetate were attenuated by vagotomy of hepatic branch and blunted by systemic treatment with capsaicin that denervates capsaicin-sensitive sensory nerves including vagal afferents. Ip injection of butyrate induced significant phosphorylation of extracellular-signal-regulated kinase 1/2, cellular activation markers, in nodose ganglia and their projection site, medial nucleus tractus solitaries. Moreover, butyrate directly interacted with single neurons isolated from nodose ganglia and induced intracellular Ca²⁺ signaling. The present results identify the vagal afferent as the novel pathway through which exogenous SCFAs execute the remote control of feeding behavior and possibly other brain functions. Vagal afferents might participate in suppression of feeding by intestine-born SCFAs.     

Coassimilation of dietary fat and benzo(a)pyrene in the small intestine: an absorption model using the killifish

Benzo(a)pyrene (BP) was dissolved in dietary fat and fed in a single dose to killifish (Fundulus heteroclitus). Fluorescence microscopic examinations of small intestinal content and frozen sections of whole small intestine revealed that during fat digestion BP was codispersed in liquid crystalline product phases produced during lipolysis (1979. Patton, J. S., and M. C. Carey, Science. 204: 145-148) and then coabsorbed with dietary lipid followed by its reappearance in intracellular fat droplets. During the time that the absorbed fat remained in the enterocytes, BP fluorescence was initially concentrated in the intracellular fat droplets and then spread throughout the cytosol of the enterocytes. Tissue analyses showed that BP was rapidly metabolized in the intestine and transported to the gallbladder. These studies show that separation of a dissolved hydrophobic carcinogen from dietary fat occurs primarily after the fat has been digested, dispersed, absorbed, and reassembled in the enterocyte. The inability of the enterocyte to discriminate between dietary fat and dissolved carcinogenic compounds may be a partial explanation of the observed link between high fat diets and the incidence of some cancers. In vertebrates, the intestine and not the liver, appears to be the major site of metabolism of dietary polycyclic aromatic hydrocarbons (PAHs).     

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.     

Responses of celiac and cervical vagal afferents to infusions of lipids in the jejunum or ileum of the rat

Multiunit celiac and single-unit cervical recordings of vagal afferents were performed before and during infusions of fatty acids, triglycerides, or saline into either the ileum or jejunum of the rat. In multiunit recordings, lipids increased activity of vagal afferents to a greater extent than saline. The greatest increases in vagal afferent activity resulted from infusions of linoleic acid, conjugated linoleic acid, or oleic acid. The triglycerides, corn oil or Intralipid, were less effective than the fatty acids in affecting vagal afferent activity. Ileal pretreatment with the hydrophobic surfactant Pluronic L-81 significantly attenuated the response of celiac vagal afferents to ileal infusion of linoleic acid. Single-unit recordings of cervical vagal afferents supported the multiunit data in showing lipid-induced increased vagal afferent activity in approximately 50% of ileal units sampled and 100% of a limited number of jejunal units sampled. These data demonstrate that free fatty acids can activate ileal and jejunal vagal afferents in the rat, and this effect can be attenuated by pretreatment with a chylomicron inhibitor. These data are consistent with the view that lipid-induced activation of vagal afferents could be a potential substrate for the inhibitory effects of intestinal lipids on gastrointestinal function, food intake, and body weight gain.     

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.     

The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)‐rich lipoprotein] assembly and secretion. The accumulation of circulating intestine‐derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose‐gradient centrifugation from differentiated Caco‐2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD‐associated TAG mobilization. Results. Among the 105 proteins identified in the CLD fraction by LC‐MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte‐specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl‐CoA synthetases) and for TAG hydrolysis. In differentiated Caco‐2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3‐β‐hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA‐IV (apolipoprotein A‐IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA‐IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high‐fat diet. Conclusions. We report for the first time the protein endowment of Caco‐2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell‐specific manner.     

The common hepatic branch of the vagus is not required to mediate the glycemic and food intake suppressive effects of glucagon-like-peptide-1

The incretin and food intake suppressive effects of intraperitoneally administered glucagon-like peptide-1 (GLP-1) involve activation of GLP-1 receptors (GLP-1R) expressed on vagal afferent fiber terminals. Central nervous system processing of GLP-1R-driven vagal afferents results in satiation signaling and enhanced insulin secretion from pancreatic-projecting vagal efferents. As the vast majority of endogenous GLP-1 is released from intestinal l-cells following ingestion, it stands to reason that paracrine GLP-1 signaling, activating adjacent GLP-1R expressed on vagal afferent fibers of gastrointestinal origin, contributes to glycemic and food intake control. However, systemic GLP-1R-mediated control of glycemia is currently attributed to endocrine action involving GLP-1R expressed in the hepatoportal bed on terminals of the common hepatic branch of the vagus (CHB). Here, we examine the hypothesis that activation of GLP-1R expressed on the CHB is not required for GLP-1's glycemic and intake suppressive effects, but rather paracrine signaling on non-CHB vagal afferents is required to mediate GLP-1's effects. Selective CHB ablation (CHBX), complete subdiaphragmatic vagal deafferentation (SDA), and surgical control rats received an oral glucose tolerance test (2.0 g glucose/kg) 10 min after an intraperitoneal injection of the GLP-1R antagonist, exendin-(9-39) (Ex-9; 0.5 mg/kg) or vehicle. CHBX and control rats showed comparable increases in blood glucose following blockade of GLP-1R by Ex-9, whereas SDA rats failed to show a GLP-1R-mediated incretin response. Furthermore, GLP-1(7-36) (0.5 mg/kg ip) produced a comparable suppression of 1-h 25% glucose intake in both CHBX and control rats, whereas intake suppression in SDA rats was blunted. These findings support the hypothesis that systemic GLP-1R mediation of glycemic control and food intake suppression involves paracrine-like signaling on GLP-1R expressed on vagal afferent fibers of gastrointestinal origin but does not require the CHB.     

Dietary diacylglycerol suppresses high fat diet-induced hepatic fat accumulation and microsomal triacylglycerol transfer protein activity in rats

We have recently shown that the long-term ingestion of dietary diacylglycerol (DAG) mainly containing 1,3-isoform reduces body fat accumulation in humans as compared to triacylglycerol (TAG) with the same fatty acid composition. The fat reduction in this human experiment was most pronounced in visceral fat and hepatic fat. Recent animal studies have also indicated that dietary DAG induces alteration of lipid metabolism in the rat liver. In the present study, the dietary effects of DAG on high fat diet-induced hepatic fat accumulation and hepatic microsomal triglyceride transfer protein (MTP) activity were examined in comparison with those of TAG diet in rats. When the TAG oil content was increased from 10 to 30 g/100 g diet, hepatic TAG concentration, hepatic MTP activity and MTP large subunit mRNA levels were significantly increased after 21 days. However, when the dietary TAG oil (30 g/100 g diet) was replaced with the same concentration of DAG oil with the same fatty acid composition, the increase of the TAG concentration and the MTP activity in the liver were significantly less and the mRNA levels remained unchanged. The MTP activity levels correlated significantly with hepatic TAG concentration.These results showed that dietary DAG may suppress high fat diet-induced MTP activity in the liver, and indicated the possibility that hepatic TAG concentration may regulate hepatic MTP activity.     

Mechanism of oleoylethanolamide on fatty acid uptake in small intestine after food intake and body weight reduction

The increase in the prevalence of human obesity highlights the need to identify molecular and cellular mechanisms involved in control of feeding and energy balance. Oleoylethanolamide (OEA), an endogenous lipid produced primarily in the small intestine, has been identified to play an important role in the regulation of animal food intake and body weight. Previous studies indicated that OEA activates peroxisome proliferator-activated receptor-alpha, which is required to mediate the effects of appetite suppression, reduces blood lipid levels, and enhances peripheral fatty acid catabolism. However, the effect of OEA on enterocyte function is unclear. In this study, we have examined the effect of OEA on intestinal fatty acid uptake and FAT/CD36 expression in vivo and in vitro. We intraperitoneally administered OEA to rats and examined FAT/CD36 mRNA level and fatty acid uptake in enterocytes isolated from the proximal small intestine, as well as in adipocytes. Our results indicate that OEA treatment significantly increased FAT/CD36 mRNA expression in intestinal mucosa and isolated jejunal enterocytes. In addition, we also found that OEA treatment significantly increases fatty acid uptake in isolated enterocytes in vitro. These results suggest that in addition to appetite regulation, OEA may regulate body weight by altered peripheral lipid metabolism, including increased lipolysis in adipocytes and enhanced fatty acid uptake in enterocytes, both in conjunction with increased expression of FAT/CD36. This study may have important implications in understanding the mechanism of OEA in the regulation of fatty acid absorption in human physiological and pathophysiological conditions.     

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.     

Vagal afferent responses to fatty acids of different chain length in the rat

The role of cholecystokinin (CCK) in the effect of dietary lipid on proximal gastrointestinal function and satiety is controversial. Recent work suggests that fatty acid chain length may be a determining factor. We investigated the mechanism by which long- and short-chain fatty acids activate jejunal afferent nerves in rats. Whole mesenteric afferent nerve discharge was recorded in anaesthetized male Wistar rats during luminal perfusion of saline, sodium oleate, and sodium butyrate (both 10 mM). Both fatty acids evoked characteristic afferent nerve responses, distinct from the mechanical response to saline, that were abolished in rats following chronic subdiaphragmatic vagotomy. The effect of oleate was abolished by the CCK-A receptor antagonist Devazepide (0.5 mg/kg), whereas the effect of butyrate persisted despite pretreatment with either Devazepide or a combination of the calcium channel inhibitors nifedipine (1 mg/kg) and the omega-conotoxins GVIA and SVIB (each 25 microg/kg). In summary, long- and short-chain fatty acids activate intestinal vagal afferents by different mechanisms; oleate acts via a CCK-mediated mechanism and butyrate appears to have a direct effect on afferent terminals.     

Sn-2-monoacylglycerol, not glycerol, is preferentially utilised for triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.) intestine

Pathways of lipid resynthesis in the intestine of fish are relatively unknown. Various reports have suggested the existence of both sn-1,3-specific (pancreatic) and non-specific (bile salt-activated) lipase activity operating on dietary triacylglycerol (TAG) in the intestinal lumen of fish during digestion. Thus, sn-2-monoacylglycerol (2-MAG) and glycerol, respective hydrolytic products of each lipase, are absorbed and utilised for glycerolipid synthesis in enterocytes via two alternative routes: monoacylglycerol (MAG) and glycerol-3-phosphate (G3P) pathways. Despite different precursors, both pathways converge at the production of sn-1,2-diacylglycerol (1,2-DAG) where TAG or phosphatidylcholine (PC) synthesis can occur. To elucidate the relative activities of MAG and G3P pathways in Atlantic salmon enterocytes, intestinal segments were mounted in Ussing chambers where equimolar mixtures of sn-2-oleoyl-[1,2,3-(3)H]glycerol (2-MAG) and [(14)C(U)]glycerol, plus unlabelled 16:0 and 18:2n-6 as exogenous fatty acid sources, were delivered in bile salt-containing Ringer solution to the mucosa. The MAG pathway predominated, over the G3P pathway, synthesizing ca. 95% of total TAG and ca. 80% of total PC after a 3 h incubation period at 10 degrees C. Further, the 1,2-DAG branch point into TAG or PC was polarised towards TAG synthesis (6:1) via the MAG pathway but more evenly distributed between TAG and PC (1:1) via the G3P pathway. Effect of long-chain saturated, monounsaturated and polyunsaturated fatty acids on the synthesized TAG/PC ratio was assessed by individually exchanging 16:0, 18:1n-9 or 18:2n-6, for 16:0+18:2n-6, in mucosal solutions. TAG synthesis was influenced considerably more than PC synthesis, via either pathway, by exogenous fatty acids utilised. 18:1n-9 significantly stimulated TAG synthesis via the MAG pathway yielding a TAG/PC ratio of 12:1. Alternatively, 18:2n-6 stimulated TAG synthesis the most via the G3P pathway (TAG/PC=4:1). 16:0 significantly attenuated TAG synthesis via either pathway. Micellar fatty acid species also significantly affected intestinal active transport mechanisms as shown by decreasing transepithelial potential (TEP) and short-circuit current (SSC) with increasing fatty acid unsaturation. The epithelial integrity was, however, not compromised after 3 h of exposure to any of the fatty acids. The implications of these findings on dietary fatty acid composition and enterocytic lipid droplet accumulation are discussed.     

Fatty acid utilisation and metabolism in caecal enterocytes of rainbow trout (Oncorhynchus mykiss) fed dietary fish or copepod oil

A combined fatty acid metabolism assay was employed to determine fatty acid uptake and relative utilisation in enterocytes isolated from the pyloric caeca of rainbow trout. In addition, the effect of a diet high in long-chain monoenoic fatty alcohols present as wax esters in oil derived from Calanus finmarchicus, compared to a standard fish oil diet, on caecal enterocyte fatty acid metabolism was investigated. The diets were fed for 8 weeks before caecal enterocytes from each dietary group were isolated and incubated with [1-14C]fatty acids: 16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:1n-9, 20:4n-6, 20:5n-3, and 22:6n-3. Uptake was measured over 2 h with relative utilisation of different [1-14C]fatty acids calculated as a percentage of uptake. Differences in uptake were observed, with 18:1n-9 and 18:2n-6 showing the highest rates. Esterification into cellular lipids was highest with 16:0 and C18 fatty acids, accounting for over one-third of total uptake, through predominant incorporation in triacylglycerol (TAG). The overall utilisation of fatty acids in phospholipid synthesis was low, but highest with 16:0, the most prevalent fatty acid recovered in intracellular phosphatidylcholine (PC) and phosphatidylinositol (PI), although exported PC exhibited higher proportions of C20/C22 polyunsaturated fatty acids (PUFA). Other than 16:0, incorporation into PC and PI was highest with C20/C22 PUFA and 20:4n-6 respectively. Recovery of labelled 18:1n-9 in exported TAG was 3-fold greater than any other fatty acid which could be due to multiple esterification on the glycerol 'backbone' and/or increased export. Approximately 20-40% of fatty acids taken up were beta-oxidised, and was highest with 20:4n-6. Oxidation of 20:5n-3 and 22:6n-3 was also surprisingly high, although 22:6n-3 oxidation was mainly attributed to retroconversion to 20:5n-3. Metabolic modification of fatty acids by elongation-desaturation was generally low at <10% of [1-14C]fatty acid uptake. Dietary copepod oil had generally little effect on fatty acid metabolism in enterocytes, although it stimulated the elongation and desaturation of 16:0 and elongation of 18:1n-9, with radioactivity recovered in longer n-9 monoenes. The monoenoic fatty acid, 20:1n-9, abundant in copepod oil as the homologous alcohol, was poorly utilised with 80% of uptake remaining unesterified in the enterocyte. However, the fatty acid composition of pyloric caeca was not influenced by dietary copepod oil.     

Retinoblastoma protein (pRb), but not p107 or p130, is required for maintenance of enterocyte quiescence and differentiation in small intestine

The function of retinoblastoma protein (pRb) in the regulation of small intestine epithelial cell homeostasis has been challenged by several groups using various promoter-based Cre transgenic mouse lines. Interestingly, different pRb deletion systems yield dramatically disparate small intestinal phenotypes. These findings confound the function of pRb in this dynamic tissue. In this study, Villin-Cre transgenic mice were crossed with Rb (flox/flox) mice to conditionally delete pRb protein in small intestine enterocytes. We discovered a novel hyperplasia phenotype as well as ectopic cell cycle reentry within villus enterocytes in the small intestine. This phenotype was not seen in other pRb family member (p107 or p130) null mice. Using a newly developed crypt/villus isolation method, we uncovered that expression of pRb was undetectable, whereas proliferating cell nuclear antigen, p107, cyclin E, cyclin D3, Cdk2, and Cdc2 were dramatically increased in pRb-deficient villus cells. Cyclin A, cyclin D1, cyclin D2, and Cdk4/6 expression was not affected by absent pRb expression. pRb-deficient villus cells appeared capable of progressing to mitosis but with higher rates of apoptosis. However, the cycling villus enterocytes were not completely differentiated as gauged by significant reduction of intestinal fatty acid-binding protein expression. In summary, pRb, but not p107 or p130, is required for maintaining the postmitotic villus cell in quiescence, governing the expression of cell cycle regulatory proteins, and completing of absorptive enterocyte differentiation in the small intestine.     

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.     

Intracellular events in the assembly of chylomicrons in rabbit enterocytes

The aim of this study was to determine the intracellular events in chylomicron assembly in adult villus enterocytes. We have used novel methods for separation of the intracellular components of the secretory compartment [rough and smooth endoplasmic reticulum (RER and SER, respectively) and Golgi], and their membrane and luminal components, from villus enterocytes isolated from rabbit small intestine. The steady state composition of the components of the secretory compartment and the intracellular pools of newly synthesized apolipoprotein B-48 (apoB-48) and triacylglycerol (TAG) was determined. The observations indicate that the SER is the main site of TAG synthesis and of chylomicron assembly. Newly synthesized apoB-48 and TAG accumulate in the SER membrane and are transferred into the lumen in a microsomal triglyceride transfer protein-dependent step. In enterocytes isolated from chow-fed rabbits, in which fat absorption is relatively slow, transfer of apoB-48 and TAG from the SER membrane into the lumen appears to be rate limiting. In enterocytes from fat-fed rabbits, TAG accumulates in the lumen of the SER, suggesting that movement out of the SER lumen becomes rate limiting, when chylomicron secretion is markedly stimulated. In these cells, the cytosolic TAG also increased to 450 microgram/g enterocytes, compared with 12 microgram/g enterocytes from chow-fed rabbits, indicating that transfer of TAG from the SER membrane into the secretory pathway can become saturated, so that newly synthesized TAG moves into the cytosol.     

Vitamin A metabolism in the human intestinal Caco-2 cell line

The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitization of cardiac vagal afferent reflexes at the sensory receptor level: an overview

The gain or sensitivity of reflexes originating in cardiac sensory receptors with vagal afferent pathways is highly dynamic. This modulation is usually attributed to central nervous system or efferent mechanisms. This paper briefly reviews evidence that modulation of reflexes originating in the heart can also occur at the sensory or afferent level. Five examples are cited: calcium antagonists, cardiac glycosides, arginine vasopressin, atrial natriuretic peptides, and changes in dietary sodium. These examples emphasize the role of ionic and humoral factors in regulation of cardiac vagal afferent function. This concept of sensory modulation of cardiac vagal afferents has implications for cardiovascular pharmacology and for pathophysiological states such as heart failure and hypertension.