Cécile
Vors
ab,
Perrine
Capolino
cd,
Clémence
Guérin
cd,
Emmanuelle
Meugnier
b,
Sandra
Pesenti
b,
Marie-Agnès
Chauvin
b,
Julien
Monteil
e,
Noël
Peretti
af,
Maud
Cansell
e,
Frédéric
Carrière
c and
Marie-Caroline
Michalski
*ab
aINRA USC1235, CarMeN laboratory, INSA-Lyon, IMBL, Villeurbanne, F-69621, France. E-mail: marie-caroline.michalski@insa-lyon.fr; Fax: +33 4 72 43 85 24; Tel: +33 4 72 43 85 70
bINSERM U1060, INRA USC1235, CarMeN laboratory, Lyon University, Univ Lyon-1, Oullins, F-69921, France
cCNRS-Aix-Marseille Université-Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, Marseille, F-13402, France
dGERME S.A., Marseille, F-13014, France
eUniv. Bordeaux, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), CNRS, UMR 5248, IPB, F- 33600, Pessac, France
fHospices Civils de Lyon, CRNH-RA, CENS, Lyon, F-69008, France
First published on 21st February 2012
There is a growing interest in the optimization of dietary emulsions for monitoring postprandial lipid metabolism in the frame of preventing metabolic diseases. Using various emulsions, we investigated in a systematic scheme the combination of (i) in vitro gastrointestinal lipolysis and (ii) absorption and metabolism of lipolysis media in Caco-2 cells. Four emulsions based on either milk fat olein (OL) or rapeseed oil (RA) as the dispersed phase and either lecithin (LE) or sodium caseinate (CA) as the emulsifier were tested. After a sequential incubation of these emulsions with gastric and pancreatic enzymes, lipolysis media were incubated with Caco-2 cells, after dilution (1:20) to maintain the barrier integrity. Both gastric and duodenal lipolysis levels were similar to values reported in vivo and the rates of lipolysis were higher with LE-stabilized emulsions than with CA-stabilized emulsions (P < 0.05). TAG secretion by Caco-2 cells was found to be higher using (i) duodenal vs. gastric media (P < 0.001) and (ii) emulsions stabilized with CA vs. LE (P < 0.01). Consistently, gene expression of both FABP2 and FATP4 induced by the duodenal media was (i) higher than that with gastric media (P < 0.001) and (ii) faster than that with model mixed micelles. Using gastric media, TAG secretion of Caco-2 cells after 12 h was higher with RA than with OL (P < 0.001). Moreover, the RA–CA emulsion increased the size of secreted lipoprotein particles (514 nm vs. 61 to 130 nm; P < 0.01). In conclusion, it was possible to observe distinct responses in the lipid metabolism of Caco-2 cells incubated with lipolysis media obtained from different dietary emulsions digested by gastrointestinal lipases in vitro.
Therefore, it now becomes important to better understand the effect of emulsion structure on lipolysis and absorption steps, considering that little information is available about the effect of oil and surfactant types.16,17 It is necessary to develop in vitro models to rapidly test the digestion and intestinal absorption of emulsions in physiological-like conditions. Meanwhile, the concept of bioavailability involves the fact that only a proportion of the nutrient provided by food is made available to target tissues after ingestion of that food. Therefore, bioavailability is influenced by the different stages of lipid metabolism: enzymatic digestion (the so-called gastrointestinal lipolysis), intestinal absorption, nutrient delivery and use by target tissues. Functional foods are meant to improve a target function or to reduce the risk of developing a pathology. Thus, a primary concern is the control of the metabolic fate of TAG by selecting their form of presentation.
In this context, the objective of the present study was two-fold. First, we studied the influence of the physical form (two types of emulsion) of two dietary lipid sources with different fatty acid compositions on the lipolysis kinetics by lipases of the gastrointestinal tract. Second, using Caco-2 cells, we investigated the intestinal absorption and the lipid metabolism in intestinal cells of the lipolysis products to test the technical feasibility of the model and possible limitations.
All chemicals, bovine bile and porcine pancreatic extract (PPE) were purchased from Sigma–Aldrich–Fluka Chemistry (St-Quentin-Fallavier, France) except the internal standard (IS; CholE1 = O-cholesteryl ethyleneglycol) used for the analysis of lipolysis products by thin-layer chromatography coupled with flame ionization detection (TLC-FID) kindly provided by Dr Dominique Lafont (ICBMS Lyon, France).
Rabbit gastric extract (RGE) was produced by GERME S.A. company (Marseille, France).
All components of mixed micelles, i.e. oleic acid, 2-oleyglycerol, soybean lecithin (phosphatidylcholine), sodium taurocholate, L-α-lysophosphatidylcholine (lyso-PC) from egg yolk and cholesterol were purchased from Sigma–Aldrich–Fluka Chemistry (St-Quentin-Fallavier, France).
Emulsion | RA–CA | OL–CA | RA–LE | OL–LE |
---|---|---|---|---|
Oil type | Rapeseed oil | Olein fraction of milk fat | Rapeseed oil | Olein fraction of milk fat |
Surfactant type | Sodium caseinate | Sodium caseinate | Soybean lecithinb | Soybean lecithin |
d 43, μm | 9.1 ± 0.8 | 8.8 ± 0.4 | 8.6 ± 0.4 | 9.1 ± 0.1 |
S, m2 g−1 | 0.78 ± 0.05 | 0.79 ± 0.02 | 1.07 ± 0.06 | 1.15 ± 0.03 |
a Values are mean ± SD, n = 3. b Soybean lecithin contained 13.2% PC, 29.6% PE, 24.6% PI, 23% Lyso-phospholipids. | ||||
FA profile (%) | ||||
4:0 to 12:0 | 0 | 18 | 0 | 18 |
14:0 | 0 | 12 | 0 | 12 |
14:1 | 0 | 1 | 0 | 1 |
16:0 | 5 | 23 | 5 | 23 |
16:1 | 0 | 2 | 0 | 2 |
18:0 | 2 | 7 | 2 | 7 |
18:1 | 62 | 28 | 62 | 28 |
18:2 n = 6 | 20 | 3 | 20 | 3 |
18:3 n = 3 | 8 | 2 | 8 | 2 |
20:0 | 0 | 3 | 0 | 3 |
20:1 | 1 | 0 | 1 | 0 |
The solution mimicking human gastric juice was prepared by mixing 21 mg of RGE powder (containing 1.41% w/w of RGL) with 3 mL of 10 mmol L−1 MES buffer (2-(N-morpholino)ethanesulfonic acid) and 150 mmol L−1 NaCl buffer, pH 6.0, in order to obtain the gastric lipase (GL) mean concentration of 100 μg mL−1 found in human gastric juice.20,21 The GL solution was prepared freshly on the day of the experiments.
The RGL activity was measured at pH 5.5 under GL standard assay conditions using tributyrin as substrate.19
The final PPL and bile salt concentrations in the incubation vessel were 250 μg mL−1 and 4.4 mmol L−1, respectively, during the simulation of the duodenal phase of digestion. It is important for the bile salt concentration to be greater than the critical micellar concentration (1–2 mmol L−1), as observed in intestinal contents.
To minimize bacterial contamination as required for further incubation of Caco-2 cells with lipolysis media, all enzyme and buffer solutions were filtered on 0.22 μm size cut-off membranes. All glass vessels and measuring instruments were washed with alcohol.
Each experiment was performed in a 50 mL thermostated vessel (37 °C), equipped with a pH electrode, and a 1-cm magnetic bar for gentle stirring at 1000 rpm. At time zero, a precise amount of emulsion (1 g for RA–CA and OL–CA; 2 g for RA–LE and OL–LE) was mixed with sterilized water to obtain a total volume of 15 mL so that the same amounts of TAG was provided in all experiments (0.7 g per 15 mL of total volume for each emulsion). Three mL of GL solution was then added and the pH was adjusted at 5.5 to simulate the gastric phase of lipolysis. At t = 30 min, 9.1 mL of pancreatic juice–bile solution were added to the reaction mixture, the pH was adjusted to 6.25 and the reaction went on for 60 additional minutes to simulate the duodenal phase of lipolysis.
The following samples were taken for analysis: (i) 1-mL samples from the reaction mixture were collected immediately at time 0 and then at times 15, 29, 40, 50, 60 and 90 min for total lipid extraction and analysis; (ii) additional samples (6 × 0.5 mL in 2-mL sterile cryotubes) of the reaction mixture were taken at times 29, 60 and 90 min, and were immediately frozen in liquid nitrogen for storage at −80 °C for later incubation with Caco-2 cells.
Each experiment was performed in duplicate. Before each experiment, GL and PL activities were measured by the pH-stat technique using previously published methods19,23 in order to verify the stability of enzymes.
Prior to the start of treatment, Caco-2 cells were incubated for 24 h with serum-free complete medium in both compartments. On the experiment day, monolayers were incubated in the apical compartment with emulsion lipolysis media diluted (1:20, targeted in preliminary experiments to avoid toxicity while still presenting significant lipid content) in serum-free complete medium. Additional experiments were performed by incubating cells with mixed oleic acid micelles whose preparation was described previously.24 Each plate contained a control sample with DMEM medium only. All solutions were maintained at 37 °C before use. The basolateral compartment received 2.5 mL of serum free complete medium.
The basolateral media were collected after incubations during 4, 8 and 12 h and were stored at −80 °C in sterile tubes for lipid analysis. After the same incubation times, cell lysates were obtained by rinsing cell layers twice with ice-cold phosphate-buffered saline (PBS) (2.7 mmol L−1 KCl, 137 mmol L−1 NaCl in 10 mmol L−1 phosphate buffer, pH 7.5), scraping the cells into 1 mL of TRI Reagent (Ambion/Applied Biosystems, Courtaboeuf, France), freezing and storing at −80 °C until genomic analysis. The experiments were repeated five times for each lipolysis product formulation.
The hydrodynamic diameter of lipoproteins secreted by Caco-2 cells was measured by photon correlation spectroscopy using a Malvern Zetasizer NanoS (Malvern, UK). Samples were concentrated by centrifugation at 13 000 rpm for 4 h at 10 °C; supernatants were analysed at 37 °C. The viscosity and the refractive index of the DMEM medium at 37 °C were respectively 0.84 cP and 1.445.
Fig. 1 Lipolysis levels of the different emulsions tested: (○) Free fatty acids (FFA) expressed as a percentage of total FA; (●) FFA and MAG expressed as a percentage of total FA. Values are means ± SD, n = 3. |
Emulsion | Gastric lipolysis (t = 29 min) | Duodenal lipolysis (t = 90 minb) |
---|---|---|
a Values are mean ± SD, n = 3. b 30 min of gastric-like digestion followed by 60 min of duodenal-like digestion. c P < 0.05 vs. OL–CA, Student's t-test. | ||
%FFA in total FA | ||
RA–CA | 4.0 ± 4.0 | 29.1 ± 2.2 |
OL–CA | 4.8 ± 1.6 | 20.9 ± 2.6 |
RA–LE | 8.4 ± 0.9 | 43.2c ± 3.6 |
OL–LE | 6.2 ± 0.6 | 42.0c ± 2.3 |
Emulsion | Lipolysis Time, min | Concentration, mmol L−1 | |||
---|---|---|---|---|---|
TAG | FFA | DAG | MAG | ||
a Gastric-like phase. b 30 min gastric-like phase followed by duodenal-like phase. | |||||
RA–CA | 29a | 26.0 | 7.4 | 4.0 | 0.0 |
60b | 7.1 | 10.9 | 6.2 | 2.3 | |
90b | 8.5 | 16.8 | 8.2 | 3.7 | |
OL–CA | 29 | 18.1 | 5.1 | 5.1 | 0.0 |
60 | 7.2 | 7.3 | 6.0 | 3.0 | |
90 | 6.2 | 8.9 | 7.8 | 5.5 | |
RA–LE | 29 | 22.2 | 8.6 | 8.6 | 0.0 |
60 | 5.7 | 26.7 | 5.7 | 5.9 | |
90 | 3.8 | 27.8 | 5.7 | 8.5 | |
OL–LE | 29 | 15.6 | 3.2 | 3.2 | 0.0 |
60 | 3.2 | 19.7 | 4.0 | 5.4 | |
90 | 2.4 | 19.3 | 4.8 | 7.2 |
From these data, the TAG conversion rate into DAG and MAG was calculated (Fig. 2). Considering the lipolysis levels obtained up to 90 min of in vitro lipolysis, the data fit a model where TAG was ultimately converted into MAG and FFA without further MAG hydrolysis at this stage (i.e., maximum lipolysis rate of 66.6% within the explored time range). Here again, we observed the enhanced susceptibility to lipolysis of lecithin-covered emulsion droplets compared with caseinate-covered droplets, regardless of the intradroplet oil composition.
Fig. 2 Triacylglycerol (TAG) conversion into diacylglycerols (DAG) and monoacylglycerols (MAG), represented as percentage of total glycerol esterified onto TAG (■), DAG (●) and MAG (○) during lipolysis. Polynomial trend curves are to guide the eye regarding the kinetics of each species appearance and/or disappearance. Note that 66% on the x-axis represent the highest theoretical lipolysis rate (2 FFA released from 1 TAG). |
Fig. 3 Effect of apical incubation with different lipolysis media (dilution 1:20 in DMEM) on Caco-2 monolayer integrity, expressed as % of initial TEER (transepithelial electrical resistance). (A) Lipolysis media after 29 min of gastric-like digestion; (B) lipolysis media after 60 min of in vitro digestion (30 min of gastric-like digestion followed by 30 min of duodenal-like digestion). (□) RA–CA (rapeseed oil emulsion stabilized with sodium caseinate); (■) OL–CA (olein fraction of milk fat emulsion stabilized with sodium caseinate); (○) RA–LE (rapeseed oil emulsion stabilized with soybean lecithin); (●) OL--LE (olein fraction of milk fat emulsion stabilized with soybean lecithin). Data are means ± SEM, n = 5. |
Incubation medium | Concentration, mmol L−1 | |||
---|---|---|---|---|
TAG | FFA | DAG | MAG | |
a Incubation media obtained by 29 min in vitro gastric-like digestion, thus devoid of bile salts (dilution 1:20). b Incubation media obtained by 30 min in vitro gastric-like digestion followed by 30 min of duodenal-like digestion of emulsions (dilution 1:20), containing 0.22 mmol L−1 of bile salts. Mixed micelles contain 5 mmol L−1 sodium taurocholate only. c Ref. 49. d Ref. 50. e Ref. 51. In each study, oleic acid was used as FFA. | ||||
29 min lipolysisa | ||||
RA–CA | 1.30 | 0.37 | 0.20 | 0 |
OL–CA | 0.91 | 0.25 | 0.25 | 0 |
RA–LE | 1.11 | 0.43 | 0.43 | 0 |
OL–LE | 0.78 | 0.16 | 0.16 | 0 |
60 min lipolysisb | ||||
RA–CA | 0.36 | 0.55 | 0.31 | 0.12 |
OL–CA | 0.36 | 0.37 | 0.30 | 0.15 |
RA–LE | 0.29 | 1.33 | 0.29 | 0.29 |
OL–LE | 0.16 | 0.99 | 0.20 | 0.27 |
Mixed micelles | ||||
This study | 0 | 0.5 | 0 | 0.2 |
Literature | 0c,d,e | 0.6c; 0.5d; 0.1e | 0c,d,e | 0.2c; 0.03d; 0.1e |
Fig. 4 Triacylglycerols (TAG) secreted by Caco-2 cells after 4 h incubation with different lipolysis media. (□) Lipolysis media after 29 min of gastric-like digestion; (■) lipolysis media after 60 min of in vitro digestion (30 min of gastric-like digestion followed by 30 min of duodenal-like digestion). Data are means ± SEM, n = 5 per treatment. Blank was 2.8 ± 1 mmol L−1. Insert shows results of multivariate ANOVA analysis. |
Considering the observed differences in TAG secretion, we further explored the impact of the different lipolysis media on the expression of genes related to lipid transport and secretion in Caco-2 cells. Fig. 5 shows the expression of genes coding for fatty acid transporters (FABP2, FATP4) and microsomal triglyceride transfer protein implicated in chylomicron assembly (MTTP), after incubation for 4 h with duodenal vs. 4 h to 8 h with gastric media from the different emulsions. In addition, we compared the gene expression from digestion media with that obtained by incubation of Caco-2 cells with model mixed micelles incubated during 4 and 8 h as usual in the literature. We also measured the gene expression after 12 h of incubation with gastric media, but the results were similar to those obtained after 8 h of incubation regardless of the gene (results not shown).
Fig. 5 FABP2, FATP4 and MTTP expressions in Caco-2 cells. Left part: after 4 and 8 h incubation with lipolysis media obtained after 29 min of gastric-like digestion and with mixed micelles. Right part: after 4 h incubation with lipolysis media obtained after 60 min of in vitro digestion (30 min of gastric-like digestion followed by 30 min of duodenal-like digestion). Data are expressed as fold-change vs. blank condition devoid of lipids and are reported as means ± SEM, n = 5 per treatment. Inserts show significant differences observed by multivariate ANOVA analysis (left, gastric media: effect of incubation time, emulsion composition and comparison with micelles; middle, for both gastric and duodenal phases: effect of emulsion composition; right, duodenal media: effect of emulsion composition and comparison with gastric media and with micelles). $P < 0.05 vs. RA–LE; §P < 0.05 vs. OL–CA and micelles; #P < 0.1 vs. other emulsions (one-way ANOVA). (□) RA–CA (rapeseed oil droplets covered with sodium caseinate); () OL–CA (olein fraction of milk fat droplets covered with sodium caseinate); () RA–LE (rapeseed oil droplets covered with soybean lecithin); () OL–LE (olein fraction of milk fat droplets covered with soybean lecithin); (■) mixed micelles. |
Regarding FABP2, the gene expression tended to be enhanced with RA–CA and RA–LE compared with OL–CA and OL–LE (P < 0.06). FATP4 expression increased with gastric media between 4 and 8 h (Fig. 5; P < 0.05). Duodenal lipolysis media induced increased gene expression of lipid metabolism compared with gastric lipolysis media for both FABP2 and FATP4 (P < 0.001). Most interestingly, incubation with duodenal media induced significant increase of fold-change after 4 h incubation for fatty acid transporters, while at least 8 h of incubation with model mixed micelles were necessary to observe the same effect on gene expression for FABP2 (P < 0.05) and FATP4 (P < 0.001).
However, regarding MTTP expression, no difference was observed among the different emulsions. Mixed micelles with taurocholate were more efficient in inducing MTTP than gastric media, devoid of bile salts. However, duodenal media were not more active than mixed micelles regarding MTTP gene expression.
Fig. 6 Lipid secretion by Caco-2 cells after incubation with lipolysis media obtained after 29 min of gastric-like digestion. (A) Triacylglycerols (TAG): (□) RA–CA; (■) OL–CA; (○) RA–LE; (●) OL–LE. Insert shows results of multivariate ANOVA analysis with repeated measurements over incubation time. *P < 0.05 vs. other emulsions, **P < 0.01 vs. other emulsions, #P < 0.05 vs. OL–LE, ##P < 0.01 vs. OL–LE. (B) Corresponding size of lipoproteins secreted after 12h. **P < 0.01 vs. other emulsions. Data are means ± SEM, n = 5 per treatment. |
Noticeably, regardless of the oil type, both emulsions stabilized with sodium caseinate underwent similar gastric and duodenal lipolysis kinetics (Fig. 1, RA–CA and OL–CA). This was also the case for both emulsions stabilized with lecithin (Fig. 1, RA–LE and OL–LE). The surfactant type appeared to have a significant effect on the lipolysis levels. Gastric and duodenal lipolysis levels were higher for lecithin-stabilized emulsions than for caseinate-stabilized emulsions (Table 2). Lecithin-stabilized emulsion thus appeared to be better substrates for lipolytic enzymes than sodium caseinate-stabilized emulsions, even though their interfacial area tended to be lower (Table 1). This result is consistent with a previous study showing that oil-in-water emulsions stabilized with lecithin resulted in pancreatic lipolysis levels a few % greater than emulsions stabilized with caseinate using an in vitro model devoid of gastric lipase.33 This observation was interpreted considering that the lipid droplets size in simulated intestinal media was larger in presence of caseinate than in presence of lyso-lecithin;34 differences in flocculation and surface charges were also observed.35 However, the differences we observed in the % of FFA released depending on the emulsifier type were greater using the present and more realistic sequential lipolysis model including gastric and intestinal steps. This result is also consistent with the report that human milk fat globules, of ∼3.5 μm and covered by a phospholipid membrane,36,37 are more efficiently lipolyzed than submicronic fat droplets of infant formula covered with milk proteins.38,39 In cow milk, despite their larger interfacial area, homogenized milk fat droplets are not more efficiently lipolyzed than natural milk fat globules (covered with a phospholipid membrane), possibly because their interface composed mainly of milk proteins slows down lipolytic activity.40In vitro, these emulsion types are usually poor substrates for pancreatic lipase alone and the preliminary action of gastric lipase is necessary for promoting lipolysis.41,42 The differences observed in the lipolysis of caseinate-stabilized and lecithin-stabilized emulsions might therefore mainly result from distinct effects of caseinate and lecithin on gastric lipase activity. Although gastric lipase is more surface-active than pancreatic lipase43 and can adsorb/penetrate at lipid-water interfaces covered by various amphiphiles such as proteins and lecithins, the activity of gastric lipase was shown to be dependent on the nature of these amphiphiles and on interfacial tension.44 Lecithins were found to increase gastric lipase activity on triglyceride emulsions.44 With caseinate-stabilized, the oil/water interfacial tension is ∼12–14 mN m−1 whereas the optimum activity of gastric lipase is recorded at ∼10 mN m−1.44 One can therefore assume that lecithin-stabilized emulsions will be better substrates for gastric lipase activity than caseinate-satbilized emulsions.
Interestingly, the lipolysis level observed in the duodenal phase in our in vitro study was similar to what observed in vivo in humans after ingestion of a coarse emulsion stabilized with both milk proteins and soybean lecithin.14 Moreover, the greater lipolysis activity observed with lecithin vs. caseinate supports the clinical data obtained in a recent human study, in which higher postprandial plasma TAG levels were observed after consumption of egg lecithin-stabilized emulsion compared with those recorded with an emulsion stabilized by caseinate + monoacylglycerols.9 Conversely to the effect of emulsifier type, we observed no significant effect of the oil FA composition (rapeseed vs. milk fat olein) on lipolysis kinetics. This result is different from some reports indicating that the specificity is higher for medium chain fatty triglycerides compared with long-chain triglycerides for bovine lipoprotein lipase45 and for pig pancreatic lipase.46 Importantly, it confirms however that both gastric and pancreatic human lipases can release medium and long chain fatty acids at similar rates.47
Regarding the molecular species generated (Table 3), importantly, the concentrations of FFA released in our in vitro model were similar to those reported in vivo in humans, i.e., 5–20 mmol L−1.14,22,48,49 Identically, the levels of DAG (5–12 mmol L−1) and MAG (0.7–4 mmol L−1) were comparable to those reported in human duodenal fluids during digestion of 10 μm-coarse emulsion.14 The corresponding relative mass% of released FFA in neutral lipid species varied from 17 to 44% (for emulsions OL–CA and RA–LE, respectively) after 90 min of lipolysis. This is also similar to what was observed in vivo in humans at the junction between duodenum and jejunum48 and comparable to what was reported in duodenal fluid, i.e., 16–46%, during digestion of coarse emulsions with the same droplet size as our emulsions.50
In lipolysis media finally incubated onto Caco-2 cells, the concentrations of FFA and MAG in our diluted lipolysis media were in the same range as those used in model mixed micelles (up to 0.6 mmol L−1 for FFA, up to 0.2 mmol L−1 for MAG).51–53 However, the lipolysis media obtained by mimicking real digestion conditions contained TAG and DAG as well as other gastric or duodenal juice components. The latter were thus more realistic than model mixed micelles to mimic the physiological conditions.
Incubation of Caco-2 cells with duodenal-like media induced enhanced expression of fatty acid transporters (FATP4, FABP2) compared with incubation with model mixed micelles. This was however, not the case for MTTP, a gene involved in chylomicron formation. Interestingly, palmitic acid was reported to down-regulate MTTP expression in Caco-2 cells compared with oleic acid and in absence of lipid.62 It was suggested that oleic acid-rich TAG might present a better ability to form stable lipid droplets than palmitic-rich TAG, which has been shown to be a key factor in enabling core expansion of chylomicron particles in enterocytes.62 Because, in our experiments, cells were exposed to complex lipid profiles including different fatty acids, our data resulted from the combined synergistic effect of different lipids and fatty acids. This could explain the lack of enhancement of MTTP with digested emulsions compared with model mixed micelles. It appears that induction of fatty acid transporters (Fig. 5) was sufficient to provide the observed increase in TAG secretion during incubation with duodenal-like phases (Fig. 4).
We would like to emphasize that RA–CA emulsion was stimulating efficiently TAG secretion by Caco-2 cells, both after gastric and duodenal-like digestion, while this emulsion was not providing the highest lipolysis rate (Fig. 1; Table 2). Results obtained using gastric-like phases showed that it was possible to use such an in vitro model devoid of bile salts to test emulsions.
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