The effects of dietary oil intake and fatty acid infusions on the expression of intestinal and liver fatty acid-binding proteins (I-FABP and L-FABP, respectively) were investigated in the small intestine of mice. A daily force-feeding for 7 days with 0.2 ml sunflower oil specifically increased L-FABP mRNA and protein levels in duodenum and proximal jejunum. This upregulation was mediated in time- and dose-dependent manners by a minute quantity of linoleic acid, the main fatty acid found in sunflower oil. The L-FABP induction was only found with long-chain fatty acids, with the nonmetabolizable, substituted fatty acid alpha-bromopalmitate being far more active. A hormonally mediated effect is unlikely because long-chain fatty acids induced L-FABP mRNA in the Caco-2 cell line cultured in serum-free medium. Therefore, long-chain fatty acids are strong inducers of L-FABP gene expression in the small intestine. In contrast to data found in the rat, I-FABP gene expression appears to be unaffected by a lipid-enriched diet in the mouse.
Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (α, γ and δ) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARα-null mice, we have found that PPARα in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARδ/α agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARα-null mice. These findings demonstrate that PPARδ can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARδ target gene in the small intestine. We propose that PPARδ contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.
Ileal bile acid-binding protein (I-BABP) is a cytosolic protein that binds bile acid (BA) specifically. In the ileum, it is thought to be implied in their enterohepatic circulation. Because the fecal excretion of BA represents the main physiological way of elimination for cholesterol (CS), the I-BABP gene could have a major function in CS homeostasis. Therefore, the I-BABP gene expression might be controlled by CS. I-BABP mRNA levels were significatively increased when the human enterocyte-like CaCo-2 cells were CS-deprived and repressed when CS were added to the medium. A highly conserved sterol regularory element-like sequence (SRE) and a putative GC box were found in human I-BABP gene promoter. Different constructs of human I-BABP promoter, cloned upstream of a chloramphenicol acetyltransferase (CAT) reporter gene, have been used in transfections studies. CAT activity of the wild type promoter was increased in presence of CS-deprived medium, and conversely, decreased by a CS-supplemented medium. The inductive effect of CS depletion was fully abolished when the putative SRE sequence and/or GC box were mutated or deleted. Co-transfections experiments with the mature isoforms of human sterol responsive element-binding proteins (SREBPs) and Sp1 demonstrate that the CS-mediated regulation of I-BABP gene was dependent of these transcriptional factors. Paradoxically, mice subjected to a standard chow supplemented with 2% CS for 14 days exhibited a significant rise in both I-BABP and SREBP1c mRNA levels. We show that in vivo, this up-regulation could be explained by a recently described regulatory pathway involving a positive regulation of SREBP1c by liver-X-receptor following a high CS diet.
The effects of chronic fat overconsumption on intestinal physiology and lipid metabolism remain elusive. It is unknown whether a fat-mediated adaptation to lipid absorption takes place. To address this issue, mice fed a high-fat diet (40%, w/w) were refed or not a control diet (3%, w/w) for 3 additive weeks. Despite daily lipid intake 7.7-fold higher than in controls, fecal lipid output remained unchanged in mice fed the triglyceride (TG)-rich diet. In situ isolated jejunal loops revealed greater [1-14C]linoleic acid uptake without TG accumulation in mucosa, suggesting an increase in lipid absorption capacity. Induction both in intestinal mitotic index and in the expression of genes involved in fatty acid uptake, trafficking, and lipoprotein synthesis was found in high-fat diet mice. These changes were lipid-mediated, in that they were fully abolished in mice refed the control diet. A lipid load test performed in the presence or absence of the LPL inhibitor tyloxapol showed a sustained blood TG clearance in fat-fed mice likely attributable to intestinal modulation of LPL regulators (apolipoproteins C-II and C-III). These data demonstrate that a chronic high-fat diet greatly affects intestinal physiology and body lipid use in the mouse. The effects of chronic fat overconsumption on intestinal physiology and lipid metabolism remain elusive. It is unknown whether a fat-mediated adaptation to lipid absorption takes place. To address this issue, mice fed a high-fat diet (40%, w/w) were refed or not a control diet (3%, w/w) for 3 additive weeks. Despite daily lipid intake 7.7-fold higher than in controls, fecal lipid output remained unchanged in mice fed the triglyceride (TG)-rich diet. In situ isolated jejunal loops revealed greater [1-14C]linoleic acid uptake without TG accumulation in mucosa, suggesting an increase in lipid absorption capacity. Induction both in intestinal mitotic index and in the expression of genes involved in fatty acid uptake, trafficking, and lipoprotein synthesis was found in high-fat diet mice. These changes were lipid-mediated, in that they were fully abolished in mice refed the control diet. A lipid load test performed in the presence or absence of the LPL inhibitor tyloxapol showed a sustained blood TG clearance in fat-fed mice likely attributable to intestinal modulation of LPL regulators (apolipoproteins C-II and C-III). These data demonstrate that a chronic high-fat diet greatly affects intestinal physiology and body lipid use in the mouse. It is well established that fat overconsumption leads to obesity in a number of animal models, including mice (1West D.B. York B. Dietary fat, genetic predisposition, and obesity: lessons from animal models. Am. J. Clin. Nutr. 1998; 67 (Suppl. 3):.: 505-512Google Scholar). Although the small intestine is responsible for body fat disposal, its role in this phenomenon has been neglected. The fact that the small intestine has long been considered a simple selective barrier able to efficiently absorb dietary fat explains this paradox. However, recent insights into intestinal physiology demonstrate that triglyceride (TG) absorption is more complex than initially believed. It is now well established that several membrane and soluble lipid binding proteins (LBPs) are involved in this process (2Niot I Besnard P. Duttaroy A.K. Spener F. Intestinal fat absorption: roles of intracellular lipid-binding proteins and peroxisome proliferator-activated receptors. In Cellular Proteins and Their Fatty Acids in Health and Disease. Wiley-VCH Verlag, Germany2003: 359-381Google Scholar). By reason of their location throughout the enterocyte and their high binding affinity for long-chain fatty acids (LCFAs), LBPs are thought to play a direct or indirect role in each step of lipid absorption: uptake, trafficking, lipoprotein synthesis, and secretion. This physiological involvement has especially been highlighted by the generation of knockout mice. For instance, the invalidation of genes encoding the intestinal fatty acid binding protein (I-FABP) (3Vassileva G. Huwyler L. Poirier K. Agellon L.B. Toth M.J. The intestinal fatty acid binding protein is not essential for dietary fat absorption in mice. FASEB J. 2000; 14: 2040-2046Google Scholar), the fatty acid transporter (FAT/CD36) (4Drover V.A. Ajmal M. Nassir F. Davidson N.O. Nauli A.M. Sahoo D. Tso P. Abumrad N.A. CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood. J. Clin. Invest. 2005; 115: 1290-1297Google Scholar), and the microsomal triglyceride transfer protein (MTP) (5Xie Y. Newberry E.P. Young S.G. Robine S. Hamilton R.L. Wong J.S. Luo J. Kennedy S. Davidson N.O. Compensatory increase in hepatic lipogenesis in mice with conditional intestine-specific Mttp deficiency. J. Biol. Chem. 2006; 281: 4075-4086Google Scholar) is associated with deep alterations of postprandial triglyceridemia. It is noteworthy that the gene expression of several intestinal LBPs is upregulated by LCFA through the activation of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) (2Niot I Besnard P. Duttaroy A.K. Spener F. Intestinal fat absorption: roles of intracellular lipid-binding proteins and peroxisome proliferator-activated receptors. In Cellular Proteins and Their Fatty Acids in Health and Disease. Wiley-VCH Verlag, Germany2003: 359-381Google Scholar). Therefore, although the intestinal functions of most LBPs remain elusive, it can be hypothesized that a high fat supply triggers a coordinated change in LBP expression, increasing intestinal absorption capacity. Moreover, it is well known that the intestinal epithelium is characterized by a dramatic cellular turnover, the whole mucosa being renewed every 3 days in the mouse (6Marshman E. Booth C. Potten C.S. The intestinal epithelial stem cell. Bioessays. 2002; 24: 91-98Google Scholar). This organ exhibits a remarkable capacity to adapt its morphology to nutritional status. Fasting decreases cell proliferation, leading to a progressive atrophy of rat intestinal epithelium (7Dunel-Erb S. Chevalier C. Laurent P. Bach A. Decrock F. Le Maho Y. Restoration of the jejunal mucosa in rats refed after prolonged fasting. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 933-947Crossref PubMed Scopus (86) Google Scholar). Conversely, refeeding restores the proliferative activity, dietary lipids being the strongest stimulators of mucosal regeneration (8Buts J.P. Vijverman V. Barudi C. De Keyser N. Maldague P. Dive C. Refeeding after starvation in the rat: comparative effects of lipids, proteins and carbohydrates on jejunal and ileal mucosal adaptation. Eur. J. Clin. Invest. 1990; 20: 441-452Google Scholar). Together, these observations strongly suggest that the high TG bioavailability of gut might not be attributable to inborn properties but to acquired properties. To determine whether such a lipid-mediated adaptation exists, a chronic high-fat diet effect on absorption capacity of the small intestine was studied in mice. Because the absorption efficiency could affect intestinal TG secretion and/or clearance (4Drover V.A. Ajmal M. Nassir F. Davidson N.O. Nauli A.M. Sahoo D. Tso P. Abumrad N.A. CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood. J. Clin. Invest. 2005; 115: 1290-1297Google Scholar), lipid load tests were also performed to explore the high-fat intestinal effect on postprandial triglyceridemia. The data reported here show that the small intestine can adapt its absorption capacity to the fat content of the diet in the mouse. They provide evidence that the small intestine plays an active role in the regulation of triglyceridemia, especially during the postprandial state. French guidelines for the use and care of laboratory animals were followed. Protocols were approved by the ethics committee of the University of Burgundy. Five week old male B6D2F1 mice weighing 20–25 g were purchased from Janvier. Mice were housed in a controlled environment that provided constant temperature and humidity and a period of darkness from 6 PM to 6 AM. Mice were acclimated individually in metabolic cages and fed a semipurified control diet for 1 week (Table 1 ). Then, mice were fed for 3 weeks a semipurified control diet containing 3% lipids (w/w), a high-fat diet containing 40% lipids (w/w) (Table 1), or refed the control diet for 3 weeks after the high-fat diet. The diets were nutritionally adequate, providing for all known essential nutrient requirements.TABLE 1Composition of the experimental dietsIngredientsControl DietHigh-Fat DietCasein21.813.5Glucose + starch61.638.1Cellulose5.83.6Minerals6.84.2Vitamins10.6Lipids340Saturated fatty acids0.34Monounsaturated fatty acids1.824Polyunsaturated fatty acids0.912Values shown are in g/100 g of dry powder. Open table in a new tab Values shown are in g/100 g of dry powder. To study the fatty acid uptake capacity of small intestine, an in situ isolated jejunal loop was realized in mice subjected to a control diet, a high-fat diet, or the high-fat diet followed by the control diet. After 16 h of starvation, mice were anesthetized with isoflurane and laparotomized. A 10 cm segment of jejunum was isolated between two ligatures. The in situ isolated segment was infused with 0.5 ml of linoleic acid in 2.19 mM solution containing 10% [1-14C]linoleic acid (51 mCi/mmol; Perkin-Elmer Life Sciences, Inc.) emulsified with 10 mM taurocholic acid. After an incubation period of 5 min, the intestinal loop was removed and the intestinal lumen was collected. The mucosa from the in situ isolated jejunal segment was then scraped off. Lipids contained in lumen and mucosa were extracted by Delsal's method (9Delsal J.L. Nouveau procédé d'extraction des lipides du sérum par le méthylal. Application aux microdosages du cholestérol total, des phosphoaminolipides et des protéides. Bull. Soc. Chim. 1944; 26: 99-105Google Scholar), and radioactivity was determined by liquid scintillation. The [1-14C]linoleic acid uptake by the jejunal segment corresponds to the difference between the quantity of [1-14C]linoleic acid infused and that remaining in the intestinal lumen at the end of the experiment. A 100 μl aliquot of lipids contained in mucosa was evaporated to dryness under a nitrogen stream. Pellet was dissolved in 40 μl of chloroform, then separated by thin-monolayer chromatography in hexane-ethyl ether-glacial acetic acid (140:60:2, v/v/v). After migration, the different lipid species were identified according to standards, [3H]triolein, and [1-14C]linoleic acid. Then, the percentage of radioactivity found in TGs, fatty acids, diglycerides/monoglycerides, and phospholipids was quantified using a Berthold scanner. To identify gene expression changes in jejunal mucosa, removed according to previous procedures (10Poirier H. Niot I. Monnot M.C. Braissant O. Meunier-Durmort C. Costet P. Pineau T. Wahli W. Willson T.M. Besnard P. Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine. Biochem. J. 2001; 355: 481-488Google Scholar) in mice subjected to a high-fat diet compared with a control diet, the low-density microarray INRArray 01.4 dedicated to lipid metabolism was used (11Martin P.G. Lasserre F. Calleja C. Van Es A. Roulet A. Concordet D. Cantiello M. Barnouin R. Gauthier B. Pineau T. Transcriptional modulations by RXR agonists are only partially subordinated to PPARalpha signaling and attest additional, organ-specific, molecular cross-talks. Gene Expr. 2005; 12: 177-192Google Scholar). The full list of 320 selected cDNA probes spotted onto the INRArray 01.4 is available at www.inra.fr/Internet/Centres/toulouse/pharmacologie/lpt.htm). The two independent diet studies were analyzed (total of n = 11/group). Total RNA from jejunal mucosa extracted with Trizol reagent (Invitrogen Life Technologies) was controlled with a Bioanalyzer 2100 (Agilent Technologies, Massy, France). For each sample, 3 μg of total RNA along with a fixed amount of 12 spiked-in yeast RNAs (used for normalization) were labeled by reverse transcription with Superscript II reverse transcriptase (Invitrogen Life Technologies) in the presence of 40 μCi of [α-33P]dCTP (ICN, Orsay, France). Radiolabeled cDNA purification as well as hybridization, washing, scanning, and image analysis with the INRArray were performed according to previously described methods (11Martin P.G. Lasserre F. Calleja C. Van Es A. Roulet A. Concordet D. Cantiello M. Barnouin R. Gauthier B. Pineau T. Transcriptional modulations by RXR agonists are only partially subordinated to PPARalpha signaling and attest additional, organ-specific, molecular cross-talks. Gene Expr. 2005; 12: 177-192Google Scholar). Statistical analysis of microarray data was performed under R (www.r-project.org) using Bioconductor packages (www.bioconductor.org). Data were log-transformed and normalized by the mean log(signals) for the 12 spiked-in yeast RNAs. A total of 137 genes exhibiting log(signals) significantly above background intensities were further analyzed. An ANOVA with the experiment and diet factors was performed for each gene. Eighteen genes whose transcripts displayed a significant diet effect (P < 0.01) and exhibited at least a 1.5-fold change between the high-fat and control diets were declared differentially expressed. The expression data for these 18 genes were then transformed to Z-scores and clustered as a heat map using the Euclidean distance and the Ward criterion. Total RNA from jejunal mucosa of mice subjected to one of the three diets was extracted with Trizol Reagent (Invitrogen Life Technologies). Total RNA was denatured, subjected to electrophoresis on a 1% agarose gel, and transferred to a GeneScreen membrane (New England Nuclear) using 20-fold concentrated 3 mM NaCl and 0.3 mM trisodium citrate, pH 7 (NaCl-citrate). cDNAs of mouse fatty acid transport protein 4 (FATP-4) (12Herrmann T. Buchkremer F. Gosch I. Hall A.M. Bernlohr D.A. Stremmel W. Mouse fatty acid transport protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase. Gene. 2001; 270: 31-40Google Scholar), mouse FAT/CD36 (a gift from Dr. P. Grimaldi, Nice, France), rat intestinal and liver fatty acid binding proteins (I-FABP and L-FABP; provided by Dr. J. I. Gordon, St. Louis, MO), and mouse apolipoprotein A-IV (apoA-IV; a gift from Dr. T. Pineau, Toulouse, France) were used as probes. MTP cDNA was obtained by Superscript II reverse transcriptase (Invitrogen Life Technologies). MTP primer sequences used were described by Sellers and Shelness (13Sellers J.A. Shelness G.S. Lipoprotein assembly capacity of the mammary tumor-derived cell line C127 is due to the expression of functional microsomal triglyceride transfer protein. J. Lipid Res. 2001; 42: 1897-1904Google Scholar). The reverse transcription reaction was carried out at 48°C for 45 min followed by 95°C for 2 min. The conditions of PCR were 95°C for 30 s, 55°C for 1 min, 72°C for 1 min (35 cycles) and 72°C for 10 min. The expected size of PCR products was 699 bp for MTP. Probes were labeled with [α-32P]dCTP (3,000 Ci/mmol; Amersham) by a Megaprime kit (Amersham). A 24 residue oligonucleotide specific for 18S rRNA was used as a probe to ensure that equivalent quantities of RNA were loaded and transferred. This oligonucleotide was 5′ end-labeled with T4 polynucleotide kinase and [γ-32P]ATP (3,000 Ci/mmol; Amersham). Filters were prehybridized for 4 h and hybridized for 16 h at 42°C according to previously published procedures (14Besnard P. Mallordy A. Carlier H. Transcriptional induction of the fatty acid binding protein gene in mouse liver by bezafibrate. FEBS Lett. 1993; 327: 219-223Google Scholar). Filters were washed successively twice in 2× NaCl-citrate at room temperature, twice in 2× NaCl-citrate with 1% sodium dodecyl sulfate at 60°C for 30 min, and finally once in 0.1× NaCl-citrate at room temperature. Autoradiograms were quantified with a calibrated densitometric scanner (Bio-Rad GS-800). cDNA was reverse transcripted from 1 μg of total RNA pretreated with DNase [DNase I Amplification grade (Invitrogen Life Technologies) and Omniscript reverse transcriptase (Qiagen)]. cDNA was diluted to 25 ng/μl using sterilized water, and real-time PCR was done in duplicate with 1 μl of cDNA, 12.5 μl of SYBR Green PCR Master Mix (qPCR™ Mastermix Plus for SYBR® Green I Fluorescein; Eurogentec), 10.5 μl of distilled water, and 1 μl of forward and reverse primers (200 nM) for a final reaction volume of 25 μl. The primer sequences were as follows: apoC-II, 5′-ACTGGAGTGAGCCAGGATAG-3′ and 5′-ACATCAGGATGACCAGGAAT-3′; apoC-III, 5′-TCAGATCCCTGAAAGGCTAC-3′ and 5′-ATAGCTGGAGTTGGTTGGTC-3′. PCR was run on the iCycler iQ system (Bio-Rad Laboratories, Inc.) using the following conditions: 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 15 s and 60°C for 30 s The fluorescence measurement used to calculate threshold cycle (Ct) was made at 60°C. Quantification of data was done by the comparative ΔΔCt method (15Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001; 25: 402-408Google Scholar). After animals were euthanized, a 1 cm length of jejunum was taken from mice in each dietary group. Tissues were placed in 4% formalin overnight and incubated in 70% ethanol, in a graded series of ethanol from 80% to 100%, and finally in xylene before it was embedded in paraffin. Paraffin sections (4 μm thick) were cut perpendicularly to the mucosal surface and fixed to polylysine-coated slides. Proliferative activity was then measured by labeling of Ki-67 antigen, which is expressed in nuclei of all cells in G1, S, and G2 phases and mitosis. After removal of paraffin with xylene, sections were rehydrated in a graded series of ethanol from 100% to 95%, and endogenous peroxidase was blocked with 3‰ H2O2 in methanol. All sections were pretreated in a microwave oven in 10 mM citrate buffer at pH 6 for 10 min at 100°C. Unspecific binding sites were blocked using 10% sheep serum before an incubation of 2 h at room temperature with the primary antibody, the monoclonal mouse antibody TEC3 (M7248; DAKO). The antibody was then linked with biotinylated rat anti-mouse IgG secondary antibody (E0464; DAKO), which was then labeled with streptavidin conjugated to horseradish peroxidase (P0397; DAKO). Ki-67-positive cells were visualized by exposing the peroxidase to 3,3′-diaminobenzidine hydrochloride chromogen substrate (K3467; DAKO) followed by counterstaining in hematoxylin. Slides were washed with deionized water, dehydrated, cleared, and mounted with permanent medium (S3026; DAKO). All cells in the active phases of the cell cycle stained brown except for G0-phase cells, which remained blue. To determine mitotic activity, an average of 30 crypts were analyzed per animal. The labeling index (%) was defined as Ki-67-positive cells/total cryptic cells. Plasma samples were collected from the axilar vein on heparinized propylene tubes. Serum TG and free fatty acid concentrations were determined using a commercial kit (Biomerieux; Wako). Stool lipids were extracted from an aliquot of feces (0.75 g) collected during the last 3 days of the control diet or the high-fat diet using the methanol/chloroform method (16Folch J. Lees M Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar). Resulting solvent was transferred to a balloon, evaporated under a nitrogen stream, placed in a vacuum desiccator overnight, and weighed. The weight difference between the starting empty balloon and the balloon containing the dried lipid was the fecal lipid amount. [1-14C]oleic acid (51 mCi/mmol) was added during lipid extraction to evaluate the efficiency of the process. Lipids were extracted from mucosa and dried. Extracts were dissolved into 1% Triton X-100/chloroform, dried under vacuum, and redissolved in water for the determination of TG using a commercial kit (Biomerieux). The cellular protein content was determined by bicinchoninic acid assay (Pierce). The mucosal content of TG is expressed as mg TG/g protein. Mice were maintained on a control diet or a high-fat diet for 21 days. After a 16 h fast, mice were weighed and received an intragastric bolus of lipids (0.5 ml of oil). Blood samples were collected from the tail vein before gavage and at 0.5, 1, 2, 3, and 4 h after gavage. Plasma TG concentration was assayed using a commercial kit (Biomerieux). The same experiment was performed in mice previously subjected to an intraperitoneal injection of tyloxapol (Sigma) in saline (500 mg/kg body weight) to block LPL activity. The results are expressed as means ± SEM. The significance of differences between groups was determined by Student's t-test. To determine whether the fat content of the diet can affect the TG absorption capacity of the small intestine, B6D2F1 mice, individually housed in metabolic cages, were subjected to the control or high-fat diet (Table 1) for 3 weeks followed or not by an additive period of 3 weeks on the control diet. In this strain, the high-fat diet did not affect the body mass, the daily caloric intake being similar regardless of the diet (Table 2 ). Despite a daily lipid intake 7.7-fold higher than in controls, the lipid elimination in feces remained unchanged in mice subjected to a chronic high fat supply. These data suggest either the existence in the mouse of an innate high absorption capacity for lipids or a fat-dependent adaptation of small intestine to the lipid content of the diet in the mouse.TABLE 2Impact of a chronic high-fat diet on body mass, caloric and lipid intake, and fecal lipid excretionParametersControl Diet (3% Fat, w/w)High-Fat Diet (40% Fat, w/w)Body mass (g)Before19.97 ± 0.619.97 ± 0.70After22.96 ± 0.7822.60 ± 0.92Caloric intake (kcal/day)12.39 ± 0.9110.92 ± 0.91Lipid intake (mg/day)129.40 ± 8.59996.14 ± 73.83aP < 0.001.Total lipid content in feces (mg/day)31.95 ± 1.1430.88 ± 0.49Mice, individually housed in metabolic cages, were fed for 3 weeks either a control diet or a high-fat diet. Lipids in feces collected during the 3 last days of treatment were extracted by the method Folch, Lees, and Sloane Stanley (16Folch J. Lees M Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar). Values shown are means ± SEM (n = 5).a P < 0.001. Open table in a new tab Mice, individually housed in metabolic cages, were fed for 3 weeks either a control diet or a high-fat diet. Lipids in feces collected during the 3 last days of treatment were extracted by the method Folch, Lees, and Sloane Stanley (16Folch J. Lees M Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar). Values shown are means ± SEM (n = 5). To address this question, the impact of a chronic high-fat supply on intestinal LCFA uptake was studied using an in situ isolated intestinal loop. By keeping intact both the luminal microenvironment (i.e., the low pH microclimate of the unstirred water layer lining the enterocytes) and the enteric lymph and blood circulation, this method allows the determination of intestinal LCFA uptake in a physiological context. As shown in Fig. 1A , mice chronically subjected to a high-fat diet exhibited a higher [1-14C]linoleic acid uptake capacity than controls. The fact that this increase was fully blunted in animals refed the control diet demonstrated the link between the intestinal LCFA uptake capacity and the TG content of the diet. To further assess the metabolic fate of LCFA in enterocytes, [1-14C]linoleic acid was assayed in the intestinal mucosa at 5 min after infusion of the lipid emulsion. No difference was found regardless of the experimental conditions used (Fig. 1B). This result suggests that the greater lipid influx into enterocytes found in the high-fat-fed mice was followed by a rapid cellular output. Moreover, the chronic high-fat supply did not affect the 14C distribution into the main classes of lipids found in intestinal mucosa (Fig. 1C). The fact that radioactivity was retrieved mainly in the TG fraction confirms the physiological relevance of this model. Intestinal epithelium undergoes rapid renewal. To explore whether a change in the cell proliferation rate of intestinal cells might contribute to the fat-mediated increase in TG absorption, the jejunal mitotic index was determined using the Ki-67 antigen method. Ki-67 protein is specifically expressed in the nucleus of cells in division. As shown in Fig. 2A , the mitotic index increased significantly in the jejunum of mice chronically subjected to the high-fat diet compared with the control diet. This change was reversible, because a return to the control value was found when mice were refed the low-fat diet. Lipid-induced mitotic activity might lead to an increase in villi size and, thus, in absorptive area. The fact that the relative intestinal mass was increased by the fat content of the diet correlates quite well with this assumption (Fig. 2B). To examine whether a chronic high-fat supply affects the intestinal gene profile and thereby influences intestinal TG absorption, the low-density microassay INRArray 01.4, dedicated to genes mainly involved in lipid metabolism, was used (11Martin P.G. Lasserre F. Calleja C. Van Es A. Roulet A. Concordet D. Cantiello M. Barnouin R. Gauthier B. Pineau T. Transcriptional modulations by RXR agonists are only partially subordinated to PPARalpha signaling and attest additional, organ-specific, molecular cross-talks. Gene Expr. 2005; 12: 177-192Google Scholar). As shown in Fig. 3 , two groups of genes were easily identified. Two genes implicated in the metabolic fate of intestinal TG-rich lipoproteins, apoA-I and apoC-III, were repressed by the high-fat diet. Conversely, this diet upregulated several genes known to play a crucial role in LCFA uptake/trafficking (FATP-4, I-FABP, L-FABP) and lipoprotein synthesis (apoA-IV). This global gene analysis highlights the strong effect of dietary fat on intestinal TG metabolism. To further analyze this regulation, a kinetic study was undertaken in a larger set of prototypical genes. As shown in Fig. 4 , chronic high-fat diet led to a dramatic upregulation of FATP-4, FAT/CD36, I-FABP, L-FABP, MTP, and apoA-IV genes. This induction was rapid and maintained throughout the exposure to the high-fat diet. Except for FATP-4, a rapid return to basal values was observed as soon as mice were refed the control diet.Fig. 4.Effects of a chronic high-fat diet on intestinal mRNA levels of genes involved in lipid absorption. Mice were fed a control diet (black bars), a high-fat diet (hatched bars), or refed the control diet after the high-fat diet (white bars). Total RNA (50 μg) isolated from jejunum of mice after different times of treatment (3, 8, 21 days) was analyzed by Northern blotting using murine cDNAs for fatty acid transport protein 4 (FATP-4), fatty acid transporter (FAT/CD36), intestinal and liver fatty acid binding proteins (I-FABP and L-FABP), microsomal triglyceride transfer protein (MTP), and apoA-IV. Values were normalized to the 18S rRNA. Values shown are means ± SEM (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ApoC-II and apoC-III are known to be a strong activator and a strong inhibitor, respectively, of LPL, an enzyme responsible for blood chylomicron clearance (17Jong M.C. Hofker M.H. Havekes L.M. Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 472-484Google Scholar, 18Shachter N.S. Apolipoproteins C-I and C-III as important modulators of lipoprotein metabolism. Curr. Opin. Lipidol. 2001; 12: 297-304Crossref PubMed Scopus (249) Google Scholar). Because microarray analysis showed that the high-fat diet decreased intestinal apoC-III gene expression (Fig. 3), mRNA levels of apoC-II and apoC-III were assayed in jejunum by quantitative PCR. As shown in Fig. 5A , a huge increase in apoC-II mRNA levels was found in mice fed the TG-rich diet, whereas apoC-III mRNA levels decreased according to the microarray analysis. A return to control values was observed in mice refed standard laboratory chow, demonstrating that this gene regulation is adaptive. Because chylomicrons can also be enriched in apoC by exchanges with hepatic lipoproteins, apoC-II and apoC-III gene expression was next determined in liver, but no change was found (Fig. 5B). A high apoC-II/apoC-III ratio in chylomicrons is expected to increase their clearance from blood and, thereby, decrease postprandial triglyceridemia. To explore this assumption, plasma TG levels were assayed in overnight-fasted mice force-fed 0.5 ml of oil. Mice subjected to the high-fat diet displayed plasma TG levels lower than mice fed the control diet (Fig. 6A ). This finding might be attributable to a decrease in intestinal TG release and/or an increase in blood TG hydrolysis by peripheral tissues. To address this question, the LPL inhibitor tyloxapol was injected intraperitoneally at 30 min before the intragastric lipid load was administered (0.5 ml). In contrast to the previous experiment, plasma TG levels from
Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (α, γ and δ) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARα-null mice, we have found that PPARα in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARδ/α agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARα-null mice. These findings demonstrate that PPARδ can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARδ target gene in the small intestine. We propose that PPARδ contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.
