Luminal lipid regulates CD36 levels and downstream signaling to stimulate chylomicron synthesis

Abstract

The membrane glycoprotein CD36 binds nano-molar concentrations of long-chain fatty acids (LCFA) and is highly expressed on the luminal surface of enterocytes. CD36 deficiency reduces chylomicron production through unknown mechanisms. In this report, we provide novel insights into some of the underlying mechanisms. Our in vivo data demonstrate that CD36 gene deletion in mice does not affect LCFA uptake and subsequent esterification into triglycerides by the intestinal mucosa exposed to the micellar LCFA concentrations prevailing in the intestine. In rodents, the CD36 protein disappears early from the luminal side of intestinal villi during the post-prandial period but only when the diet contains lipids. This drop is significant 1 h after a lipid supply and associates with ubiquitination of CD36. Using Chinese hamster ovary cells (CHO) expressing CD36, it is shown that the digestion products, LCFA and diglycerides, trigger CD36 ubiquitination. In vivo treatment with the proteasome inhibitor MG132 prevents the lipid-mediated degradation of CD36. In vivo and ex vivo, CD36 is shown to be required for lipid activation of extracellular signal-regulated kinase 1/2 (ERK1/2) which associates with an increase of the key chylomicron synthesis proteins, apolipoprotein B48 (ApoB48) and microsomal triglyceride transfer protein (MTP). Therefore, intestinal CD36 possibly through ERK1/2 mediated signaling is involved in the adaptation of enterocyte metabolism to the post-prandial lipid challenge by promoting the production of large triglyceride-rich lipoproteins that are rapidly cleared in the blood. This suggests that CD36 may be a therapeutic target for reducing the post-prandial hypertriglyceridemia and associated cardiovascular risks.

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LUMINAL LIPID REGULATES CD36 LEVELS AND DOWNSTREAM SIGNALING TO
STIMULATE CHYLOMICRON SYNTHESIS
Thi Thu Trang Tran1, Hélène Poirier
Pelsers2, Valérie Petit1, Pascal Degrace4, Marie-Claude Monnot
Abumrad3, Philippe Besnard
1, Lionel Clément
1, Fatiha Nassir3, Maurice M.A.L.
1, Jan F.C. Glatz2, Nada A
1 and Isabelle Niot1

Physiologie de la Nutrition, U 866 INSERM/Université de Bourgogne, AgroSup Dijon, Dijon,
France
Maastricht, The Netherlands2, Department of Medicine, Center for Human Nutrition, Washington
University, St Louis, MO 63110, USA3, Physiopathologie des Dyslipidémies, U 866
INSERM/Université de Bourgogne, Dijon, France4.
Running head: Lipid signaling by intestinal CD36 and chylomicron formation
Address correspondence to: Isabelle Niot, PhD, Pr, Physiologie de la Nutrition, AgroSup Dijon, 1
Esplanade Erasme, 21000 Dijon. Tel: +33 3 80 77 40 24. E-mail: niot@u-bourgogne.fr

The membrane glycoprotein CD36 binds
nano-molar concentrations of long-chain
fatty acids (LCFA) and is highly expressed
on the luminal surface of enterocytes. CD36
deficiency reduces chylomicron production
through unknown mechanisms. In this
report, we provide novel insights into some
of the underlying mechanisms. Our in vivo
data demonstrate that CD36 gene deletion
in mice does not affect LCFA uptake and
subsequent esterification into triglycerides
by the intestinal mucosa exposed to the
micellar LCFA concentrations prevailing in
the intestine. In rodents, the CD36 protein
disappears early from the luminal side of
intestinal villi during the post-prandial
period but only when the diet contains
lipids. This drop is significant 1 h after a
lipid supply and associates with
ubiquitination of CD36. Using Chinese
hamster ovary cells (CHO) expressing
CD36, it is shown that the digestion
products, LCFA and diglycerides, trigger
CD36 ubiquitination. In vivo treatment with
the proteasome inhibitor MG132 prevents
the lipid-mediated degradation of CD36. In
vivo and ex vivo, CD36 is shown to be
required for lipid activation of extracellular
signal-regulated kinase 1/2 (ERK1/2) which
associates with an increase of the key
chylomicron synthesis proteins,
apolipoprotein B48 (ApoB48) and
microsomal triglyceride transfer protein
(MTP). Therefore, intestinal CD36 possibly
through ERK1/2 mediated signaling is
involved in the adaptation of enterocyte
metabolism to the post-prandial lipid
challenge by promoting the production of
large triglyceride-rich lipoproteins that are
1, Department of Molecular Genetics, Maastricht University, P.O. Box 616, 6200 MD
rapidly cleared in the blood. This suggests
that CD36 may be a therapeutic target for
reducing the
hypertriglyceridemia
cardiovascular risks.

CD36 (also known as fatty acid translocase,
FAT) is a transmembrane glycoprotein
expressed in many
multifunctional protein homologous to the
class B scavenger receptor SR-B1. CD36
facilitates uptake of long chain fatty acids
(LCFA) in cardiomyocytes (1) and adipocytes
(2-3) and that of oxidized-LDL (oxLDL) by
macrophages (4). CD36 is involved in platelet
aggregation by binding thrombospondin and
collagen (5), phagocytosis of apoptotic cells
by macrophages (6) and in the cyto-adhesion
of erythrocytes infected with Plasmodium
falciparum (7). In addition, CD36 has recently
been shown to play a role in taste-perception
of dietary fatty acid on the tongue by
triggering a cell signaling cascade (8-10).
Deletion of CD36 in mice highlighted
importance of this protein for optimal
utilization of dietary lipids. Significant
impairment in uptake of LCFA by skeletal
muscle, heart and adipose tissues was shown
(2). Insulin- and
translocation of CD36 from an intracellular
pool to the sarcolemna was documented and
postulated to increase the muscle efficiency by
allowing adaptive changes in LCFA uptake
and utilization (11-12).
deficient mice exhibit loss of the spontaneous
preference for lipid-rich foods and a decrease
of orosensory-mediated rise in digestive
secretions (8-9). In humans, variants in the
CD36 gene have been associated with
post-prandial
associated and
tissues. It is a
exercise-dependent
Finally, CD36

http://www.jbc.org/cgi/doi/10.1074/jbc.M111.233551The latest version is at
JBC Papers in Press. Published on May 24, 2011 as Manuscript M111.233551

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abnormalities of lipid and glucose metabolism
(13) and with altered susceptibility to the
metabolic syndrome (14) and diabetes
associated coronary disease (15).
In the small intestine, expression of CD36 is
especially high in the brush border membrane
(BBM) of duodeno-jejunal enterocytes both in
rodents and humans (16-17). Although this
location correlates with the main site of LCFA
absorption, the physiological role played by
CD36 in the gut remains incompletely
understood. In isolated enterocytes from the
proximal intestine, CD36 deletion impairs
LCFA uptake (18). However, in vivo, the lack
of impairment in intestinal net lipid absorption
in CD36 (-/-) mice (18-19) as well as in
subjects with type I CD36 deficiency (20)
seems to preclude a quantitative role of
intestinal CD36 in LCFA uptake similar to
that demonstrated in skeletal muscle, heart and
adipose tissues. By contrast, lipoprotein
synthesis and/or secretion are deeply impaired
in enterocytes from CD36 (-/-) mice where
smaller chylomicrons are produced. This event
might explain
hypertriglyceridemia found in CD36-deficient
mice and human (20-22). Indeed, the
efficiency of blood triglyceride (TG) clearance
is positively linked to chylomicron size (23).
Therefore, it can be speculated that CD36
plays a role in regulating the metabolic fate of
lipids rather than LCFA uptake in the small
intestine. However, the molecular mechanisms
by which this effect would occur remain
unknown. One possibility could be a CD36
dependent modulation of Extracellular signal-
Regulated Kinase (ERK) since it was
previously shown to regulate lipoprotein
formation (24-26) and it is activated by ligand
binding to CD36 (27).
To explore this concept, experiments were
conducted in vivo in rats and in wild-type or
CD36 (-/-) mice, ex vivo in intestinal segments
and in vitro in stably transfected Chinese
hamster ovary (CHO) cells. The present data
show that CD36
ubiquitination and degradation triggered by
digestion products during intestinal lipid
absorption. This process is associated with
CD36 dependent activation of ERK and with
increased expression of the key proteins of
lipoprotein synthesis ApoB48 and MTP.

Experimental procedures
the postprandial
undergoes rapid
Materials- Antibodies against rat-CD36 were
generated in rabbits and used as previously
described (16). Anti-mouse CD36 (R&D System
or Santa Cruz Biotechnology), mouse anti-
ubiquitin (Zymed Laboratories, Invitrogen),
Anti-Phospho-ERK1/2 (Thr202/Tyr204) and
Anti-ERK1/2 (Cell Signaling Technology), anti-
ApoB, anti-HSC70 (Santacruz Biotechnology),
anti-MTP (BD Sciences), secondary antibody
peroxidase conjugate:
(Chemicon International),
(Sigma Aldrich), donkey anti-goat (Santacruz
Biotechnology) were from commercial sources.
Protein A/G Plus-Agarose Immunoprecipitation
Reagent was from Santa Cruz, MG132 (Z-leu-
leu-leu-al) from Sigma Aldrich, the enhanced
chemiluminescence (ECL)
PerkinElmer, triolein from Sigma and oleic acid,
2-monolein, or miolein from Larodan Fine
Chemicals, Sweden.
Animals and experimental procedures- Care
and use of laboratory animals followed
guidelines of the Animal Ethic committee of
Burgundy University, which approved all
protocols. Rats or mice were housed in a
controlled environment (constant temperature
and humidity, darkness from 6 PM to 6 AM).
They were fed a standard laboratory chow
containing 3% (w/w) lipids (UAR A-04 for rats
and mice, Usine d’Alimentation Rationnelle).
The alipidic diet was prepared by Safe, France.
Rats: Male Wistar rats weighting 180-200g
(Elevage Dépré, France) were fasted for 48 h. In
a first protocol, animals were refed either a
standard laboratory chow containing 3% lipids
(wt/wt, sunflower oil) or a fat-free diet. After
sacrifice (by isoflurane anesthesia), jejunal
samples (1 cm) taken 24 cm after the pylorus
were prepared for immunohistochemistry using
antibody against CD36 raised in rabbits (16).
In a second protocol, 48 h fasted rats were force-
fed 3 ml sunflower oil or sucrose (isocaloric
5.27 M). Mucosa from jejunal segments (25 cm
taken 10 cm after pylorus) was obtained at
different times after and used to prepare
homogenates and BBM. Mucosa samples were
rapidly frozen and stored at –80 °C until RNA
purification.
Mice: Male C57Bl6 (CD36 (+/+) and CD36 (-/-)
mice (provided by Dr Jan Glatz) (12 weeks of
age) weighing 25-30 g, were fasted for 12 h
prior to oil gavage (0.5 ml of sunflower oil) and
sacrificed by isoflurane anesthesia. The small
intestine was divided into three equal parts. The
first cm was taken from the mid part of the small
goat
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anti-rabbit
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intestine, considered as the jejunum and treated
for immohistochemistry using antibody anti-
CD36 from R&D system. The mucosa from
jejunal segments was scrapped at 1 h, 2 h and 4
h after the gavage for homogenate preparation
and mRNA analysis. The same protocol was
performed in mice subjected 30 min before the
gavage to an intraperitoneal injection of 100 µl
Dimethyl sulfoxide (DMSO) containing 14
mg/kg body weight of the proteasome inhibitor
MG132 while the control group received the
same volume of DMSO. The animals were
sacrificed 4 h after the oil gavage.
Jejunal homogenate and brush border
membrane- Scrapped mucosa obtained from rats
were weighed and homogenized (1 g /16.7 ml of
100 mM mannitol, 10 mM Tris-HCl, pH 7.1 =
buffer A) using a Dounce potter homogenizer.
Purified BBM were prepared according to
Shirazi-Beechey et al. (28). Briefly, MgCl2 was
slowly added to homogenates to reach a final
concentration of 10 mM. The samples were
stored on ice for 30 min followed by
centrifugation (3,000 x g, 30 min, 4°C).
Supernatants were centrifuged (27,000 x g, 30
min, 4°C) (Beckman LE-70, SW41Ti rotor). The
supernatant was removed, the pellet resuspended
in 3.8 mol of buffer B (300 mM mannitol, 10
mM Tris-HCl, 0.1 mM MgSO4, pH 7.4) and
centrifuged (27,000 x g, 30 min, 4°C) (Beckman
centrifuge, 70.1Ti rotor). The supernatant was
removed, and the BBM pellet resuspended in
150 µl of buffer B and stored at –20°C. Purity of
BBM preparation and membrane yield were
evaluated by assaying the BBM specific enzyme
sucrase-isomaltase according to the Dahlqvist’s
method (29) and enzyme content was related to
1 mg of starting mucosa. The proportion of the
sucrase activity retrieved in the BBM fraction
corresponds to the efficiency of BBM extraction.
Enrichment was obtained by dividing specific
activity (UI per mg of protein) of the sucrase in
BBM fraction by that of the homogenate. Yield
and enrichment were consistent with previous
reports (28). To prepare microsomal and
cytosolic fractions, the remaining homogenate
from the same animals was centrifuged (18,000
x g, 10 min, 4°C) and the supernatant transferred
and centrifuged (105,000 x g, 60 min, 4°C), to
yield a microsomal (pellet) and a cytosolic
(supernatant) fraction. The pellet was re-
suspended in 200 µl of buffer B. The fractions
were stored at –20°C. When mice were used, a
jejunal homogenate was obtained by adding 50
mg of jejunal mucosa to 0.5 ml of buffer A with
1% triton X-100.
Jejunal triglyceride content was evaluated after
lipid extraction of mucosa with Delsal (30).
Dried extracts dissolved in 1% triton X-100 (in
chloroform) were dried then re-dissolved in
water for triglyceride determination using
CHOD-PAP (Boehringer Mannheim).
Intestinal fatty acid uptake: in situ isolated
jejunal loop- Isofluran anaesthetized, 16 h fasted
mice were laparatomized and a 10 cm jejunal
loop was isolated in situ and was infused with
0.5 ml of a radiolabeled solution of linoleic acid
in 2180 µM solution containing 10% of [1-14C]
linoleic acid (50 mCi / mmol) emulsified with 10
mM sodium taurocholate to form micelles.
Radioactivity incorporation into the different
lipid classes in the mucosa was determined (31).
Isolated intestinal segment culture- The
proximal small intestine from 16 h fasted wild
type and CD36 (-/-) mice was cut into small
segments (3 cm length) taken 3 cm after the
pylorus, split as describred by Pardo et al., (32).
After a 15 min stabilization period, intestinal
segments were treated with 240 µM linoleic acid
or vehicle (ethanol 0.01%) for 10 to 20 min. The
treatment was ended by submerging the
intestinal segment into liquid nitrogen. The
mucosa was collected by scraping into ice-cold
RIPA buffer containing anti-protease and anti-
phosphatase cocktail.
Evaluation of CD36 ubiquitination in mice
jejunum and in CHO cells- CD36 was
immunoprecipitated overnight at 4°C from 500
µg of jejunal homogenate using 1 µg of anti-
CD36 (R&D System). Then protein A/G-agarose
plus beads were added and the incubation
continued for another 2-3 h. The final pellet was
washed 3 times in RIPA buffer and boiled for
10 min in SDS sample
centrifugation, the supernatant was analyzed by
SDS-PAGE and the proteins electroblotted onto
PVDF membranes. Membranes were treated
with a denaturating buffer (6 M guanidine-HCl
20 mM Tris-HCl, pH 7.5, 5 mM beta-
mercaptoethanol, 1 mM PMSF) for 30 min at
4°C, followed by extensive PBS washing and
then used for detection of ubiquitin or CD36
proteins.
In CHO cells, the carboxyl-FLAG-tagged wild
type (WT) or carboxyl lysine mutant (K/A)
human CD36 were generated as described (33).
Cells were transfected (Lipofectamine 2000,
Invitrogen) with empty vector (control), or with
vector containing WT CD36 or CD36 where the
buffer. After
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two carboxyl terminal lysines (469 and 472)
were substituted with alanine (K/A). The cells
were then selected for stable transfections using
hygromycin. Stably transfected cells plated in 6
well plates were incubated for 2 h with either
BSA or BSA together with 100 µM of oleic acid,
triolein (Sigma), 2-monolein,
(Larodan Fine Chemicals, Sweden). The cells
were lysed, and clarified
immunoprecipitated with FLAG affinity gel
(Sigma). Immunocomplexes were eluted with
FLAG peptide, resolved by SDS-PAGE, and
immunoblotted with
antibody (Zymed) or anti-CD36 antibody
(Santa-Cruz).
Real-time PCR, Western blotting and Elisa
analyses- mRNA levels were measured by real
time PCR (9,31). CD36 protein levels were
determined using Western blotting (16) and
sandwich enzyme-linked immunoassay (ELISA)
(34). Quantification of immune signals was by
Densitometry and Quantity One software
(Biorad).
Statistics- All data are presented as means ±
SEM for the indicated number of animals.
Significance of differences between groups of
observations was tested with a paired Student’s t
test. P ≤ 0.05 was considered significant.

RESULTS

CD36 gene deletion does not affect micellar
LCFA uptake and TG re-esterification in mouse
jejunum- To determine whether CD36 plays a
critical role in intestinal LCFA uptake under
close to physiological FA delivery conditions,
emulsified lipid solution containing [1-14C]-
linoleic acid as tracer was infused into the lumen
of in situ jejunal loops isolated in fasted wild-
type and CD36 (-/-) mice. This in vivo technique
maintains both intestinal microclimate (i.e.
unstirred water layer and cell polarization) and
lymph/blood circulations (31). A large lipid load
(2180 µM) was used to mimic the post-prandial
period. Five minutes after infusion, the
distribution of radioactivity in lumen (L) and
mucosa (M) was similar in CD36 (+/+) and
CD36 (-/-) mice confirming that CD36 does not
contribute to net lipid absorption in the mouse
small intestine (Fig. 1-A). Similar results were
obtained using a lower concentration of linoleic
acid (400 µM) (data not shown). Similarly,
analysis of radioactivity incorporation into the
different lipid classes in the mucosa did not
reveal a difference in TG synthesis between
or diolein
lysates were
either anti-ubiquitin
CD36 (-/-) mice and controls (Fig. 1-B). Cellular
radioactivity was recovered mostly in TG 5 min
after infusion supporting physiological relevance
of the experimental model used.
Lipid-dependent disappearance of CD36
from the luminal side of jejunal epithelium- To
gain insight into how intestinal CD36 can
influence the metabolic fate of LCFA in
enterocytes, the effect of nutritional status on
CD36 localization in the absorptive epithelium
was explored by immunohistochemistry. In
fasted mice, prominent staining was detected in
the epithelial lining of villi (Fig. 2-A).
Surprisingly, disappearance
immunostaining was observed 4 h after an oral
lipid load. The fact that no staining was
observed in CD36 (-/-) mice confirms signal
specificity. Similar results were observed in
fasted rats refed a standard laboratory chow
containing 3% lipids (w / w). Interestingly,
immunostaining persisted when fasted rats were
fed a fat-free diet, demonstrating that CD36
removal from the apical side of enterocytes is a
physiological event related to dietary lipids.
CD36 removal from the BBM is not
associated with CD36 recovery in intracellular
fractions- To explore whether the disappearance
of staining resulted from a translocation of
CD36 from the apical plasma membrane to an
intracellular pool as reported for SR-B1 (35),
CD36 content in jejunal mucosa homogenates
and in purified BBM was assayed by sandwich
ELISA. Fasted rats were subjected to an oral
lipid load (3 ml sunflower oil) to optimize the
detection of changes in CD36 localization. To
confirm lipid dependence a set of rats was
forced-fed a sucrose solution providing a similar
caloric load to the oil gavage. Activity of
sucrase, a BBM enzyme marker, was determined
to verify the efficiency of BBM purification.
BBM fractions from fasted and force-fed rats
exhibited equivalent enrichment and degree of
purity and thus can be compared (Table 1).
Consistent with earlier data (Fig. 2-B), a lipid
load led to a 3-fold decrease of CD36 in jejunal
BBM from rats 6 h after gavage (Fig. 3-A, C).
Similar results were found when the data were
expressed as a ratio of CD36 level to sucrase
activity demonstrating it was BBM-related (Fig.
3-B). In agreement
immunohistochemistry results, this effect was
lipid-mediated since no change was observed in
sucrose force-fed animals (Fig. 3-A, B). To
explore whether lipid-mediated disappearance of
CD36 from BBM is associated with intracellular
of this
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transfer of the protein, CD36 was assayed in
jejunal homogenates by both ELISA (Fig. 3-D,
E) and Western blotting (Fig. 3-F). Total CD36
content in mucosa of lipid forced-fed rats
remained low as compared to fasted controls.
To further analyze this result, CD36 content in
cytosolic and microsomal/endosomal fractions
(105.000 x g supernatants
respectively) isolated from jejunal homogenates
was assayed by ELISA. Cytosol CD36 content
was 8-fold higher in lipid-fed than in fasted rats
(2.47 + 0.34 % of total CD36 vs 0.29 + 0.09 %,
P<0.05, n = 3). However, it remained
dramatically low and could not account for the
65 % decrease in protein in BBM fractions and
homogenates. Since no CD36 was found in
microsomal/endosomal
shown), these results strongly suggest that a
major part of internalized intestinal CD36 is
degraded 6 h after the lipid load.
Dietary lipids specifically induce a rapid
drop in jejunal CD36 content- To study the
kinetics of the lipid-mediated disappearance of
CD36 protein, CD36 content in whole mucosa
and in purified BBM was explored at various
times following the oral lipid load in rats. As
shown in Fig. 4-A, the amount of CD36 in BBM
dramatically decreased 1 h after force-feeding.
This effect remained significant 12 h after the
gavage, likely due to the high lipid load used in
this experiment. The same pattern was observed
when CD36 was assayed in the whole mucosa
which excludes quantitative CD36 accumulation
in the intracellular compartment (Fig. 4-B).
Similar data were also obtained in the mouse
(Fig. 4-C). The lipid-mediated decrease in CD36
protein in jejunal mucosa is specific to this
scavenger receptor since two other lipid-binding
proteins, co-expressed
differentiated enterocytes were either unchanged
(I-FABP: data not shown) or increased (MTP)
(Fig. 4-D). The lipid-mediated drop in intestinal
CD36 levels did not associate with a change in
CD36 mRNA 1 h after the lipid load (Fig. 4-E,
F). In contrast, a significant decrease in CD36
mRNA levels occurred 4 h after the load in rats
(Fig. 4-E) and mice (Fig. 4-F). Thus, two
regulatory mechanisms seem to be involved in
the decrease of intestinal CD36 content triggered
by dietary lipids: an early post-translational
mechanism followed by a more delayed
transcriptional regulation.
Dietary lipids trigger degradation of
intestinal CD36 via the ubiquitin-proteasome
pathway- Poly-ubiquitination tags proteins
and pellets,
fractions (data not
with CD36 in
targeted to proteasomes or lysosomes for
subsequent degradation (36). To determine if the
lipid-mediated drop in CD36 levels measured in
intestinal mucosa involves the ubiquitin-
proteasome pathway, jejunal CD36 from mice
subjected or not to a lipid load was immuno-
precipitated before immuno-blotting with an
antibody against ubiquitin. The CD36 signal
obtained at 2 h was smeared typical of poly-
ubiquitination. This smearing was diminished at
4 h after the lipid load likely reflecting
degradation of the ubiquitinated protein (Fig. 5-
A). Therefore, dietary fat induces in the small
intestine rapid ubiquitination of CD36.
Fat digestion produces 1,2-diglycerides, 2-
monoglycerides and free LCFA. To explore
which among these lipids might be responsible
for downregulation
ubiquitination of FLAG-tagged human CD36
(FLAG-CD36) stably expressed in CHO cells
was examined next. To determine whether Lys-
469 and Lys-472 in the carboxyl-cytoplasmic
tail of CD36 are the putative ubiquitination sites,
CHO cells stably expressing a FLAG-CD36
double mutant where the Lys were substituted
by Ala were used. Incubation of CHO cells for 2
h in presence of 100 µM of the various lipids
showed that oleic acid and 1,2-diolein led to
CD36 ubiquitination (Fig. 5-B, lanes 2). This
effect was dependent on presence of Lys-469
and Lys-472 since it was not observed with
mutated CD36 (Fig. 5-B, lanes 3). In contrast, 2-
monoolein and triolein were inefficient in
inducing CD36 ubiquitination. To assess
whether ubiquitination induces proteosomal
degradation of CD36, fasted mice were treated
with the proteasome inhibitor MG132 before
gavage with oil. Consistent with our earlier data
(Fig. 4-C), a dramatic drop in CD36 content in
the intestinal mucosa took place 4 h after the
gavage. MG132 treatment prevented this lipid-
mediated decrease demonstrating involvement of
the proteasome in CD36 degradation (Fig. 6-A,
B).
The lipid-mediated degradation of intestinal
CD36 affects the
extracellular signal-regulated kinase (ERK1/2),
ApoB48 and MTP protein expression- There is
evidence from the literature indicating that
ligand binding to CD36 mediates cell-specific
responses especially via activation of the
MAPKs family pathway (37) including ERK1/2
(27,38). Since the modulation of ERK1/2
activation has been demonstrated to be involved
in the assembly of ApoB containing lipoproteins
of CD36 levels,
activation levels of
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(24-25), we hypothesized that a defect in CD36
signaling could be involved in the reduction of
intestinal lipoprotein production in CD36 (-/-)
mice (21).
To examine if the lipid-mediated degradation of
CD36 observed in the postprandial period is
linked to ERK activation in vivo, the
phosphorylation of this MAPK was measured in
mice intestines after the lipid load when CD36
was degraded or not by MG132 treatment (Fig.
6-A). As shown in figure 6-B, there was basal
activation of ERK1/2 in the fasted state (control)
which was similar in CD36 (+/+) and CD36 (-/-)
mice. However in CD36 (+/+) mice, four hours
after the lipid ingestion as CD36 protein levels
significantly decreased an associated decrease in
ERK1/2 activation was observed (Fig. 6-B).
Moreover, this decrease was blunted when
degradation of CD36 was prevented by MG132.
In contrast, in CD36 (-/-) mice neither the lipid
load nor MG132 treatment significantly affected
ERK1/2 activation (Fig. 6-A, C). Together, these
in vivo data suggested that dietary LCFA
mediated ERK1/2 activation during intestinal
absorption is dependent on CD36 protein level.
This interpretation would imply that LCFA are
able to activate ERK1/2 via CD36 before its
degradation. The direct effect of LCFA on CD36
protein level and ERK activation, was
determined ex vivo, in isolated intestinal
segments from CD36 (+/+) and CD36 (-/-) mice
incubated in presence of linoleic acid (240 µM,
complexed with BSA) for a short time (10 to 20
min) to avoid CD36 degradation. As expected,
LCFA treatment led to activation of ERK1/2
after 5 min (data not shown) and 10 min (+ 60
%), when level of CD36 had not decreased yet.
After 20 min of LCFA treatment, ERK1/2
activation was reduced but at that time CD36
protein levels were significantly decreased (-30
%) (Fig. 7-A). In agreement with our in vivo
data LCFA treatment did not affect ERK1/2
activation in intestinal segments derived from
CD36 (-/-) mice (Fig. 7-A).
These data demonstrated that LCFA induce
CD36-dependent ERK1/2
subsequently CD36 degradation which correlates
with a reduction of ERK1/2 activation. Finally,
to determine whether the CD36-associated
modulation of ERK1/2 affects proteins involved
in formation of chylomicrons, ApoB48 and MTP
protein content was evaluated in mucosa of
intestinal segment isolated from CD36 (+/+) and
CD36 (-/-) mice and cultured with LCFA. Our
data demonstrated that
activation and
LCFA treatment
increased ApoB48 after 10 min when lipid
mediated activation of ERK1/2 was highest and
MTP after 20 min when ERK1/2 activation was
down-regulated in CD36 (+/+) mice segments.
These effects were not observed in CD36 (-/-)
mice segments (Fig. 7-B).
DISCUSSION
CD36 deficiency has been documented to impair
intestinal chylomicron production but how this
occurs, remains unclear. In this report, we
provide novel insight into the potential
underlying mechanisms. We present the first
demonstration that CD36 disappears from the
luminal side of intestinal villi early during the
post-prandial period. This phenomenon is lipid
dependent and is triggered by LCFA and/or
diglycerides derived from gastric and pancreatic
digestion of dietary TG. In addition it is highly
lipid sensitive and observed in both rats and
mice fed a standard chow containing only 3%
lipids. We also show that lipid-induced down-
regulation of CD36 in the intestinal mucosa
reflects targeted CD36 proteolysis by the
ubiquitin-proteasome pathway. Moreover, we
document in vivo and ex vivo that CD36 is
required for dietary-lipid activation of ERK1/2
and that the activation level parallels that of
CD36 protein. Finally, we demonstrate, using ex
vivo experiments that CD36/lipid-dependent
modulation of ERK1/2, associates a sequential
up-regulation of ApoB48 and MTP, two proteins
required for the assembly of large chylomicrons.
Indirect evidence suggesting lipid-induced
down-regulation of
previously reported when rat red quadriceps
CD36 level decreased 2 h after intravenous
infusion of a TG emulsion (39). A potential
mechanism was provided by the finding in C2C12
myotubes that LCFA can induce CD36
ubiquitination and its subsequent degradation by
the proteasome (33). Our findings indicate that
during lipid absorption, digestive products, in
particular LCFA and diglycerides induce down-
regulation of luminal CD36 content in the
intestinal mucosa via its internalization and
targeting to degradation.
CD36 protein was
This rapid degradation of CD36 in the intestine
argues against its efficient participation in LCFA
uptake during the
Consistent with this, CD36 deletion does not
affect lipid uptake (Fig. 1) using in situ isolated
intestinal segments and a micellar FA delivery
postprandial period.
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system. This model keeps intact the unstirred
water layer lining the BBM, a property of
enterocytes that is not applicable to other lipid-
utilizing cells (i.e. adipocytes, myocytes and
hepatocytes). Our data are in agreement with the
fact that fecal lipid content was not altered in the
CD36 (-/-) mice (22). Enterocytes are subjected
to very high levels of lipid during postprandial
periods, which is not the case for other cells.
Luminal lipid levels would greatly exceed CD36
capacity since the protein functions at low
(nano-molar) concentrations of LCFA. In
addition, the unstirred water layer, which has a
low pH gradient induces LCFA protonation,
which facilitates their membrane permeation (for
review see (40)). The findings in this study
together with previously published work (19)
suggest that CD36 likely functions in early
stages of absorption to initiate events that
regulate intracellular FA processing.
There is a large body of evidence to support the
function of membrane CD36 in transducing
intracellular signals after binding a number of
ligands such as thrombospondin-1 in endothelial
cells (41-42), oxLDL in macrophages (43) and
LCFA in taste bud cells (8-9). CD36 localizes to
plasma membrane detergent resistant micro-
domains implicated in cell signaling (44). CD36-
dependent signal transduction is generally
associated with activation of Src and MAP
family kinases for review see (37). The
cytoplasmic C-terminal tail of CD36 mediates
these interactions in various tissues (10,42-
43,45) including mouse taste buds (9-10).
Moreover, in macrophages it has been
demonstrated that the C-terminal domain of
CD36 is associated with MEKK2 (a MAP kinase
pathway component) (43) and CD36-dependent
activation of the ERK1/2 has been demonstrated
in at least two studies (27,38). Our data support
involvement of ERK1/2 in CD36-mediated
regulation of chylomicron formation. Indeed, in
vivo as well as ex vivo LCFA activation of
ERK1/2 is tightly correlated with CD36 protein
levels and is reduced in parallel with CD36
during lipid absorption. Furthermore CD36 is
required for LCFA activation of ERK1/2 as
shown by our in vivo and ex vivo studies using
CD36 (-/-) mice. As with numerous surface
receptors (36), exposed to persistent and high
supply of ligand, intestinal CD36 is down-
regulated by its LCFA ligands via ubiquitination
and degradation. This would serve as a feedback
regulatory and potentially protective mechanism
(36,46) .
Interestingly, the LCFA induced downregulation
may also be important for optimization of
chylomicron formation. The lipid CD36-
dependent ERK1/2 activation associated with an
early increase in intestinal ApoB48 versus a
more delayed increase in MTP. These two
proteins are essential for the assembly and
secretion of chylomicrons (40). In the small
intestine, MTP is required for transfer of the TG
to the ApoB containing prechylomicrons in the
lumen of the endoplasmic reticulum, a process
which plays a rate-limiting role in lipoprotein
synthesis. Mice with conditional intestine-
specific MTP deficiency
malabsorption, cytoplasmic
accumulation in enterocytes
disappearance of ApoB48 lipoproteins (47).
ApoB48 is the preferred protein coat for
chylomicron lipid and is needed for efficient
chylomicron formation (48). Furthermore our
data are in agreement with the lower lymphatic
Apo B48 secretion observed in CD36 (-/-) mice
(22).
MAPK activation has
implicated in regulating levels of the ApoB48
and MTP proteins. MEK1/2 inhibition (and
consequently ERK1/2 inhibition) decreased
ApoB48 production in hamster enterocytes (25).
In contrast, the ERK1/2 cascade exerts an
inhibitory effect on MTP gene transcription (26).
In the liver, inhibition
phosphorylation pathway triggers formation of
large sized ApoB lipoproteins (24). Consistent
with these findings, in our studies, MG132
treatment of CD36 (+/+) mice which prevents
the reduction in ERK1/2 phosphorylation also
prevents up-regulation of MTP mRNA (data not
shown). Together the data suggest that dietary
lipid induced CD36 degradation may play a role
in the formation of large chylomicrons via
down-regulation of ERK1/2 activation and
subsequent up-regulation of MTP.
We thus propose a model in which LCFA
binding to the extracellular domains of CD36 at
early stages of intestinal lipid absorption
activates ERK1/2 which triggers an increase in
mucosal ApoB48 protein. LCFA also induce
CD36 ubiquitination. In presence of high LCFA,
a significant proportion of CD36 is progressively
ubiquitinated and degraded leading to a decrease
in CD36 levels. Consequently, CD36 dependent
activation of intestinal ERK1/2 also decreases
which would be important to trigger the increase
in MTP protein required for ApoB48 lipidation
(24) and chylomicron formation. These CD36
exhibit
lipid
and
fat
droplet
virtual
been previously
of the ERK
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dependent events may explain why in CD36 (-/-
) mice subjected to high fat diet more intestinal
lipid retention is observed and why smaller
lipoprotein particles are secreted (21). In
conclusion, our data indicate that CD36
degradation which may be dependent on the
LCFA content of the diet could be the signal
leading to the formation of large ApoB48 rich
TG lipoproteins via reducing ERK1/2 activation
and increasing MTP level.
Overall, these novel observations suggest that
CD36 operates as a lipid sensor, responsible for
transducing signals related to the dietary lipid
content that optimize the formation of large
chylomicrons containing ApoB48.
A better understanding of the factors that
regulate CD36 and its signaling pathways and
how they contribute to lipid dependent intestinal
adaptation (31,40) is important. It might provide
new therapeutic approaches and/or dietary
recommendations that optimize chylomicron
size and consequently blood chylomicron
clearance to decrease prevalence of post-prandial
hypertriglyceridemia and the associated risk of
cardiovascular diseases.

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FOOTNOTES

Acknowledgements: This research was supported by the National Institute of Health and Medical
Research (INSERM) and the National Institute of Agronomic Research (INRA) and by grants from
the Research Program in Human Nutrition SensoFAT of the French National Research Agency
(L'Agence Nationale de la Recherche, ANR) and from the Burgundy Council (to PB) and by grants
R01 DK60022 and DK33301 from the National Institute of Diabetes and Digestive and Kidney
Diseases (to NAA).

Abbreviations: CD36: fatty acid translocase; I-FABP: intestinal fatty acid-binding protein; MTP:
microsomal triglyceride transfer protein; ApoB: apolipoprotein B; ERK1/2: extracellular signal-
regulated kinase 1/2; P-ERK1/2: Phosphorylated ERK; HSC70: Heat shock cognate protein 70;
LCFA: long-chain fatty acid; TG: triglycerides; DG: diglycerides; BBM: brush border membrane;
CHO: Chinese hamster ovary.

FIGURE LEGENDS

Fig. 1: CD36 deficiency does not alter jejunal uptake and esterification of micellar fatty acids in
the mouse.
Linoleic acid (0.5 ml, 2180 µM) in solution containing [1-14C] linoleic acid (51 mCi / mmol)
emulsified with 10 mM taurocholic acid was infused into in situ isolated jejunal loops of fasted mice.
Percentage of [1-14C] linoleic disappeared (D) (mainly absorbed or oxidized) was determined by
subtraction of the radioactivity remaining into the mucosa (M) from the radioactivity remaining into
the initial lumen (L). Values shown are means ± SEM, n = 3.
B- Relative distribution of [1-14C] radioactivity in various lipid classes in the mucosa, PC:
phophatidyl-choline; PS/PI: phophatidyl-serine / phophatidyl-inositol; PE: phophatidyl-ethanolamine;
DG/MG: di-and mono-glyceride; FFA: free fatty acid, TG: triglyceride.

Fig. 2: Lipid-dependent disappearance of CD36 protein from the luminal side of mice and rat
jejunum.
A- Immuno-localization of CD36 in jejunal epithelium in mice. Fasted controls were compared to
experimental mice 4 h after force-feeding with 0.5 ml oil. Immunostaining of CD36 on CD36 (-/-)
mice demonstrates specificity of the antibody (R&D System).
B- Immuno-localization of CD36 in jejunal epithelium in the rat. Fasted controls were compared to
rats refed for 6 h a standard laboratory chow containing 3 % lipids (w / w), or a lipid free diet.
Specific CD36 immuno-staining was performed using an antiserum raised in rabbit against purified
rat CD36 and a FITC-conjugated anti-rabbit antibody raised in goat. In negative control micrographs,
the primary antibody was omitted.

Fig. 3: CD36 removal from brush border membranes (BBM) does not quantitatively match its
intracellular transfer.
CD36 content in purified BBM and jejunal mucosa from fasted control rats and from rats 6 h after
forced-feeding with 3 ml of oil were assayed by both ELISA (A, B, D, E) and Western-blotting (C, F).
To determine whether the oil effect on CD36 levels in BBM and jejunal mucosa is specific, a set of
animals were also force-fed with a sucrose solution providing a similar caloric load as the oil gavage.
Activity of sucrase, a BBM enzyme marker, was determined to verify the efficiency of BBM
purification (B, E). Mean + SEM, n = 3. * P < 0.05; ** P< 0.01; *** P <0.001.



Fig. 4: Time course of lipid-induced drop in jejunal CD36 protein content
CD36 content in purified BBM (A) and jejunal mucosa (B) from fasted and oil gavaged rats was
assayed by ELISA at 1 h, 4 h, 6 h, 9 h and 12 h after the gavage. Expression of CD36 in mice mucosa
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was analyzed by Western-blotting (15 µg) at 1 h and 4 h after force-feeding with oil and compared to
fasted controls (C). To explore the specificity of the oil effect on CD36, expression of MTP was also
examined (D). These data were normalized to HSC70 expression, which remained unchanged in these
experimental conditions and served as an internal control for protein loading. Mean + SEM, n = 3. * P
< 0.05; ** P< 0.01.
CD36 mRNA levels were assayed in jejunal mucosa from fasted control rats and experimental
animals at 1 h, 4 h, 6 h, 12 h and 30 h after force-feeding with 3 ml of sunflower oil (E) and in jejunal
mucosa from fasted and experimental mice at 1 h or 4 h after force-feeding with 0.5 ml of oil (F).
Data were normalized to 18S rRNA for differences in RNA loading. Mean + SEM, n = 5. ** P< 0.01;
***, P < 0.001.

Fig. 5: Lipids induce poly-ubiquitination of jejunal CD36 in vivo and in CD36-transfected CHO
cells in vitro.
A- Fasted control mice were forced-fed with 0.5 ml of oil and sacrificed 2 h or 4 h later. The jejunal
mucosa homogenates were immuno-precipitated (IP) with anti-CD36 antibody. Immuno-complexes
were analyzed by Western-blotting (WB) with anti-ubiquitin or anti-CD36 antibodies. Data are
representative of 3different experiments.
B- CHO cells transfected with an empty vector (lane 1), FLAG-CD36 (lane 2), or double mutated-
CD36 (K469A and K472A) (lane 3) were treated 2 h with (100 µM) of the indicated lipid; oleic acid,
1-2 diolein, 2-monolein added in presence of BSA. The cells were lysed and clarified lysates
immunoprecipitated (IP) with FLAG affinity gel. The immuno-complexes were then analyzed by
Western blotting (WB) using an anti-ubiquitin antibody or an anti-CD36 antibody.

Fig. 6: Degradation of CD36 by the proteasome (A, B) leads to a decrease in the ERK1/2 (A, C)
activation in CD36 (+/+) mice but not in CD36 (-/-) mice.
Control fasted CD36 (+/+) and CD36 (-/-) mice were injected intraperitoneally with MG132 (14
mg/kg) 30 min before gavage (0.5 ml oil) and sacrificed 4h later.
CD36, HSC70, Phospho-ERK1/2, ERK1/2 levels in jejunal mucosa were analyzed by Western-
Blotting. A representative signal is shown from four independent experiments, quantified by
densitometry and standardized to HSC70 signal or total ERK1/2 as the loading control. Data are
expressed in percent of controls not treated with MG132. Mean + SEM, n = 4. * P < 0.05.

Fig. 7: The Long Chain Fatty Acid mediated ERK1/2 activation requires CD36 and triggers an
up-regulation of ApoB48 and MTP protein expression.
Isolated intestinal segments from in CD36 (+/+) and CD36 (-/-) mice cultured in presence of linoleic
acid (240 µM, complexed with BSA) during 10 to 20 min
A: CD36, Phospho-ERK1/2, ERK1/2 levels in jejunal mucosa were analyzed by Western-Blotting. A
representative signals is shown from four independent experiments, quantified by densitometry and
standardized to total ERK1/2 as the loading control.
B: ApoB48, MTP, HSC70 levels in jejunal mucosa were analyzed by Western-Blotting. A
representative signal is shown from four independent experiments, quantified by densitometry and
standardized to HSC70 as the loading control.
Data are expressed in percent of controls not treated with LCFA. Mean + SEM, n = 4. * P < 0.05, **
P<0.01.

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Table 1: Characteristics of Brush Border Membrane (BBM) fractions isolated from rat jejunal mucosa.
Homogenates and BBM fractions were prepared as indicated in Experimental section. Sucrase activity was
evaluated in each fraction. Five rats have been used in each group.
Total sucrase activity
(U/min/mg protein)
Enrichment factor
(BBM/homogenate)
Efficiency of
BBM extraction
(%) (%)
H Homogenatet BBM BBM
Fasted controls
0.79 ± 0.0911.53 ± 1.5214.82 ± 1.4313.44 ± 0.96
Oil load
0.50 ± 0.06 ns
9.72 ± 0.85ns
20.17 ± 2.38ns
10.51 ± 1.72ns
Sucrose load
0.66 ± 0.11ns
11.00 ± 0.42ns
16.67 ± 0.56ns
15.27 ± 0.53ns
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