The FASEB Journal • Research Communication
CD36-dependent signaling mediates fatty acid-induced
gut release of secretin and cholecystokinin
Sinju Sundaresan,* Rafiq Shahid,‡ Terrence E. Riehl,† Rashmi Chandra,‡
Fatiha Nassir,* William F. Stenson,† Rodger A. Liddle,‡ and Nada A. Abumrad*
*Department of Medicine, Center for Human Nutrition, and †Division of Gastroenterology,
Washington University School of Medicine, St. Louis, Missouri, USA; and ‡Department of Medicine,
Division of Gastroenterology, Duke University, North Carolina, USA
ABSTRACT Genetic variants in the fatty acid (FA)
translocase FAT/CD36 associate with abnormal post-
prandial lipids and influence risk for the metabolic
syndrome. CD36 is abundant on apical enterocyte
membranes in the proximal small intestine, where it
facilitates FA uptake and FA-initiated signaling. We
explored whether CD36 signaling influences FA-medi-
ated secretion of cholecystokinin (CCK) and secretin,
peptides released by enteroendocrine cells (EECs) in
the duodenum/jejunum, which regulate events impor-
tant for fat digestion and homeostasis. CD36 was
immunodetected on apical membranes of secretin- and
CCK-positive EECs and colocalized with cytosolic gran-
ules. Intragastric lipid administration to CD36ⴚ/ⴚ mice
released less secretin (ⴚ60%) and CCK (ⴚ50%) com-
pared with wild-type mice. Likewise, diminished secre-
tin and CCK responses to FA were observed with
CD36ⴚ/ⴚ intestinal segments in vitro, arguing against
influence of alterations in fat absorption. Signaling
mechanisms underlying peptide release were examined
in STC-1 cells stably expressing human CD36 or a
signaling-impaired mutant (CD36K/A). FA stimulation
of cells expressing CD36 (vs. vector or CD36K/A)
released more secretin (3.5- to 4-fold) and CCK (2- to
3-fold), generated more cAMP (2- to 2.5-fold), and
enhanced protein kinase A activation. Protein kinase A
inhibition (H-89) blunted secretin (80%) but not CCK
release, which was reduced (50%) by blocking of cal-
modulin kinase II (KN-62). Coculture of STC-1 cells
with Caco-2 cells stably expressing CD36 did not alter
secretin or CCK release, consistent with a minimal
effect of adjacent enterocytes. In summary, CD36 is a
major mediator of FA-induced release of CCK and
Abbreviations: BSA, bovine serum albumin; CaM-KII, cal-
modulin kinase II; CCK, cholecystokinin; DHA, docosa-
hexaenoic acid; DMEM, Dulbecco’s modified Eagle’s me-
dium; EEC, enteroendocrine cell; ELISA, enzyme-linked
immunosorbent assay; FA, fatty acid; FBS, fetal bovine serum;
GFP, green fluorescent protein; GPR, G-protein-coupled re-
ceptor; HBSS, Hanks’ balanced saline solution; IBMX,
3-isobutyl-1-methylxanthine; LA, linoleic acid; OA, oleic acid;
PA, palmitic acid; PKA, protein kinase A; PMSF, phenylmeth-
anesulfonyl fluoride; RIA, radioimmunoassay; SNP, single-
nucleotide polymorphism; SSO, sulfo-N-succinimidyl oleate;
WT, wild type
0892-6638/13/0027-1191 © FASEB
secretin. These peptides contribute to the role of CD36 in
fat absorption and to its pleiotropic metabolic effects.—
Sundaresan, S., Shahid, R., Riehl, T. E., Chandra, R.,
Nassir, F., Stenson, W. F., Liddle, R. A., Abumrad, N. A.
CD36-dependent signaling mediates fatty acid-induced
gut release of secretin and cholecystokinin. FASEB J. 27,
1191–1202 (2013). www.fasebj.org
Key Words: CCK 䡠 cAMP 䡠 calcium
Dietary fatty acids (FAs) trigger release of a number
of peptides, including cholecystokinin (CCK) and se-
cretin, by enteroendocrine cells (EECs) (1, 2). CCK is
secreted by I cells (3, 4) and secretin by S cells (5–7),
localized primarily to the duodenum and jejunum.
Both peptides influence fat absorption and participate
in nutrient sensing and regulation of energy balance
(1, 8, 9). CCK regulates gallbladder contraction, secre-
tions by the stomach and acinar pancreas, and colonic
motility (10). Secretin plays an important role in buff-
ering the acidic chyme in the intestinal lumen by
stimulating water and bicarbonate secretion and en-
hances CCK effects on the acinar pancreas and gall-
bladder (11). In addition, both CCK and secretin have
prosatiety effects (12, 13).
A number of cellular surface receptors for FA, includ-
ing the family of G-protein-coupled receptors (GPRs)
and the scavenger receptor CD36, have been identified.
Long-chain FAs are recognized by GPR120, GPR40, and
CD36 (14 –16). CD36 facilitates cellular FA uptake (14)
and, like the GPRs (17, 18), mediates FA-induced signal
transduction, influencing cellular calcium for release of
neurotransmitters (19) and arachidonic acid and pros-
taglandins (20). In taste cells, FA sensing by CD36
mediates fat perception and preference (21, 22) and
induces the cephalic phase of digestion (22). In hu-
mans, oral FA detection thresholds are higher in sub-
1
Correspondence: Department of Medicine, Center for
Human Nutrition, 660 S. Euclid Ave., Campus Box 8031, St.
Louis, MO 63110, USA. E-mail: nabumrad@dom.wustl.edu
doi: 10.1096/fj.12-217703
This article includes supplemental data. Please visit http://
www.fasebj.org to obtain this information.
1191
jects carrying single-nucleotide polymorphisms (SNPs)
that reduce CD36 levels (23).
CD36 and the GPRs have distinct distribution pat-
terns in the intestine. CD36 is abundant in the proxi-
mal small intestine (24, 25) on villi enterocytes, where
it contributes to FA and cholesterol uptake (25) and to
signaling events important for initiating chylomicron
formation (26 –28). The GPRs (GPR120 and GPR40)
are more abundant in distal segments, localize primar-
ily to EECs, and were shown to mediate FA-induced
peptide release (17, 29). The signaling pathways in-
volved in peptide release involve cAMP and protein
kinase A (PKA) as well as PKA-independent mecha-
nisms (30 –32). Based on its ability for FA sensing and
signal transduction, CD36 could contribute to the
regulation of EEC peptide release. However, this possi-
bility had been unexplored and was examined in the
present work. Using CD36⫺/⫺ and wild-type (WT)
mice, we documented in vivo and in vitro, using intes-
tinal segments, robust effects of CD36 on FA-induced
release of secretin and CCK. In studies using STC-1
cells, we examined the mechanisms underlying CD36-
mediated effects and demonstrated involvement of
cAMP and calcium.
MATERIALS AND METHODS
Materials
Antibodies used are listed in Table 1. FA-free bovine serum
albumin (BSA) fraction IV, PKA inhibitor H-89 dihydrochlo-
ride (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesul-
fonamide), cAMP inhibitor MDL-12,330A hydrochloride [cis-
N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine], and
calmodulin kinase inhibitor II (CaM-KII) KN-62 were from Sigma-
Aldrich (St. Louis, MO, USA).
Animals
CD36-null (CD36⫺/⫺) and WT mice on the C57BL/6
background were housed in a facility with a 12-h light-dark
cycle and fed chow ad libitum (Purina, St. Louis, MO, USA).
Female mice, 3– 4 mo old, were denied access to food for
16 h before euthanasia. Mouse care and use followed
guidelines of the animal ethics committee of Washington
University School of Medicine (St. Louis, MO, USA).
Transgenic CCK-green fluorescent protein (GFP) mice, used
TABLE 1. Antibodies
Antibodies Host
CD36 Goat
␤-Actin Mouse
Chromogranin A Rabbit
GFP Chicken
Phospho-PKA substrates (RRXS*/T*) Rabbit
PKA C-␣ Rabbit
Ran Goat
Secretin Rabbit
1192 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org
for CCK immunohistochemistry, were developed by the Mu-
tant Mouse Regional Resource Center (University of Mis-
souri, St. Louis, MO, USA). In these mice, GFP expression is
driven by the CCK promoter, allowing detection of CCK-
positive cells by immunofluorescence (33). Animals were
denied access to food for 16 h with ad libitum access to water
before the small intestine was harvested.
Evaluation of gastric emptying
Gastric emptying was measured as described previously (34).
In brief, 1 ml of phenol red (100 ␮g/ml) was administered
orally to mice after overnight food withdrawal, and stomachs
were collected 15 min later. The residual phenol (A) was
recovered by rinsing with 10 ml of Na2HPO4 solution (0.1 M),
and aliquots were diluted 1.5-fold (S1) and 5-fold (S2) before
measurement of absorbance at 570 nm (BioTek Instruments,
Winooski, VT, USA). The concentration was obtained from a
calibration curve. Dye recovered from stomachs of mice
sacrificed immediately after dye administration served as
controls (B). Percentage of gastric emptying was calculated as
100 ⫺ (A/B) ⫻ 100.
Immunohistochemistry
Mice were administered a mixture of 5-bromo-2=-deoxyuri-
dine (120 mg/kg) and 5-fluoro-2=-deoxyuridine (12 mg/kg)
intraperitoneally 90 min before sacrifice to label S-phase cells.
Proximal and distal small intestines were opened longitudi-
nally, fixed in 10% formalin, and paraffin embedded. Cut
sections (5 ␮m) were deparaffinized, followed by antigen
retrieval (99°C, 18 min) in a pressurized chamber (Biocare
Medical, Concord, CA, USA). Sections were incubated (1 h)
in donkey serum (2%) and BSA (3%) to block nonspecific
binding and then were incubated overnight (4°C) with pri-
mary antibodies, followed by fluorescently labeled (Alexa
Fluor) secondary antibodies (1 h, 1:200). Images were taken
using a Zeiss inverted fluorescent microscope (Carl Zeiss Inc.,
Thornwood, NY, USA). For colocalization, doubly positive
cells at ⫻400 were counted for 5 mice/antigen. For CCK,
immunohistochemical analysis was performed as described
previously (33).
Secretin and CCK release in vivo
After overnight food withdrawal, mice were administered an
intragastric load of olive oil or saline (16 ␮l/g body weight),
and peptide release into the blood was measured 30 min
later. CCK and secretin were extracted on C18 columns
(Waters, Milford, MA, USA) and quantified. Plasma CCK was
measured by bioassay using amylase secretion from isolated
rat pancreatic acini (35). Plasma from 3 mice was pooled to
obtain adequate sensitivity (36). Secretin was measured by
Source Dilution
R&D Systems (Minneapolis, MN, USA) 1:100
Santa Cruz Biotechnology (Santa Cruz, CA, USA) 1:10,000
Abcam (Cambridge, MA, USA 1:200
Abcam 1:1000
Cell Signaling Technology (Danvers, MA, USA) 1:1000
Cell Signaling Technology 1:1000
Santa Cruz Biotechnology 1:10,000
Phoenix Pharmaceuticals (Belmont, CA, USA) 1:1000
SUNDARESAN ET AL.
enzyme-linked immunosorbent assay (ELISA; Phoenix Phar-
maceuticals, Belmont, CA, USA).
Secretion by proximal intestinal loops
The proximal small intestine isolated from WT and CD36⫺/⫺
mice after 16 h of food withdrawal was cut into two 10-cm
segments, starting at 2 cm after the pylorus. The pieces were
filled with Hanks’ balanced saline solution (HBSS) with or
without 100 or 300 ␮M linoleic acid (LA; plus protease and
phosphatase inhibitors), tied at both ends, and incubated for
1 h at 37°C. The media were collected, lyophilized, rehy-
drated with 500 ␮l, and assayed. Secretin was measured by
ELISA, and CCK was quantified by radioimmunoassay (RIA)
using 125I-labeled CCK-8 and antisera 92128, which recognize
biologically active CCK (37).
STC-1 and Caco-2 cells
The mouse EEC line (STC-1) was cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA,
USA) with 20% fetal bovine serum (FBS). STC-1 cells stably
expressing human WT or mutated CD36 (CD36K/A) with
C-terminal lysines 469 and 472 substituted by alanine (38)
were generated by electroporation (Nucleofector Kit V;
Lonza, Cologne, Germany) followed by selection in gentami-
cin (400 ␮g/ml). Caco-2 cells expressing WT CD36 or the
mutated form CD36K/A were generated as described for
STC-1 cells and maintained in DMEM with 20% FBS.
Cocultures of enterocytes (Caco-2 cells) with EECs (STC-1
cells) were established by serial seeding. First, 3.0 ⫻ 106
differentiated Caco-2 cells expressing CD36 or empty vector
were seeded in DMEM containing 10% FBS. Twelve hours
later, 1 ml of culture medium containing 0.3 ⫻ 106 STC-1
cells, with or without CD36 expression, was added to the dish.
A 1:10 ratio of STC-1 cells to Caco-2 cells was chosen to
simulate the relative physiological distribution. One day later,
the mixed culture was washed, serum starved for 8 h, and
incubated (60 min, 37°C) in HBSS (with Ca2⫹ and Mg2⫹)
with 50 and 200 ␮M LA (plus 5 ␮M BSA) or with BSA alone
(controls). Secretin and CCK release was determined by
ELISA and RIA (37), respectively.
Peptide release by STC-1 cells
Cells (1⫻106) in 6-well plates were serum starved in DMEM
and then were stimulated (1 h) by addition of 50 –200 ␮M FA;
LA, docosahexaenoic acid (DHA), oleic acid (OA), and
palmitic acid (PA) complexed to 5 ␮M BSA were added in
HBSS (no glucose) containing aprotinin (200 kallikrein-
inhibiting units/ml). Media were collected for secretin anal-
yses by ELISA. Cells were lysed for protein assays in buffer [20
mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM Na2EDTA; 1 mM
EGTA; 1% Triton X-100; 2.5 mM sodium pyrophosphate; 1
mM glycerol 2-phosphate; 1 mM Na3VO4; 1 ␮g/ml leupeptin;
and 1 mM phenylmethanesulfonyl fluoride (PMSF)] contain-
ing a protease inhibitor mix (Roche Diagnostics, Inc., India-
napolis, IN, USA). CCK release was measured by RIA (37).
Measurement of cAMP, PKA activation, and
intracellular Ca2ⴙ
Intracellular cAMP was measured using the DetectX Direct
High Sensitivity Cyclic AMP Chemiluminescent Immunoassay
Kit (Arbor Assays, Ann Arbor, MI, USA). In brief, serum-
starved STC-1 cells (5⫻105/well) were stimulated with FA
(100 ␮M) complexed with BSA (5 ␮M) or BSA alone (30 min,
CD36 MEDIATES RELEASE OF GUT PEPTIDES
37°C) in HBSS containing 1 mM 3-isobutyl-1-methylxanthine
(IBMX). Cellular protein was determined in lysates from
duplicate wells (DC Protein Assay; Bio-Rad Laboratories,
Hercules, CA, USA). PKA activation was determined from
phosphorylation of PKA substrates (with the RRXS/T motif)
and nuclear content of the catalytic PKA subunit PKA-C␣.
Intracellular calcium was measured as described previously
(20), using Fura-2/AM dye (2.5 ␮M).
Western blot analyses
Protein signals were detected using the Odyssey Infrared
System (Li-Cor Biosciences, Lincoln, NE, USA) as described
previously (20). Cell proteins separated and transferred to
polyvinylidene fluoride membranes were blocked and incu-
bated overnight with primary antibodies (4°C) and then for 1
h with infrared dye-labeled secondary antibodies (room tem-
perature).
Statistical analyses
Statistical analyses were performed with GraphPad Prism 4
software (GraphPad Software Inc., San Diego, CA, USA).
Differences obtained by 1-way ANOVA were considered sig-
nificant at values of P ⱕ 0.05. The Bonferroni test was
performed to identify groups that were different.
RESULTS
CD36⫺/⫺ mice show reduced levels of intestinal pep-
tides in response to a lipid load: CD36 is abundant in
the proximal intestine, where it has been shown to
facilitate uptake of FA and cholesterol (25) and to
promote chylomicron formation (26). We examined
the influence of CD36 deletion on fat-induced secre-
tion of CCK and secretin, peptides with important roles
in fat absorption that are released by EECs localized
primarily in proximal segments (3–7). Plasma CCK and
secretin levels were measured in WT and CD36⫺/⫺
mice 30 min after an intragastric load of olive oil.
CD36⫺/⫺ mice had 50% lower CCK (Fig. 1A) and 60%
lower secretin levels (Fig. 1B) than WT mice. Peptide
levels were similar in saline-administered WT and
CD36⫺/⫺ mice.
CD36 deficiency alters gut fat absorption by impair-
ing chylomicron formation and shifting more luminal
fat to distal parts of the small intestine (26, 28). These
changes might contribute to the reduction of fat-
induced CCK and secretin release in CD36⫺/⫺ mice by
altering fat exposure of EECs in the proximal part of
the intestine. We first compared gastric emptying rates
in WT and CD36⫺/⫺ mice and found no significant
differences between the two groups (68.45⫾4.34 vs.
71.32⫾6.39, n⫽4). To directly examine the role of
CD36 in FA-induced release of secretin and CCK inde-
pendent of fat absorption, intestinal segments were
isolated from WT and CD36⫺/⫺ mice and tested for
FA-induced peptide release. Secretin and CCK release
were similar in WT and CD36⫺/⫺ segments under basal
conditions (data not shown). Addition of 100 and 300
␮M LA enhanced CCK release from WT segments by
2.3- and 3.6-fold above basal. No enhancement was
1193
Figure 1. CCK and secretin release in vivo or in vitro by incubated intestinal segments from WT and CD36⫺/⫺ mice. A,
B) In vivo. After overnight food withdrawal, mice were administered an intragastric load of olive oil or saline (16 ␮l/g body
weight), and peptide release into the blood was measured 30 min later. CCK (A) and secretin (B) were extracted on C18
columns and quantified by bioassay or ELISA, respectively. All assays were performed on plasma pooled from 3 mice per
genotype per treatment. Data are means ⫾ se [n of pooled (3/pool) samples⫽3]. C, D) Intestinal segments in vitro.
Proximal intestinal segments (10-cm length, beginning at 2 cm after the pylorus) isolated from mice after 16 h of food
withdrawal, were filled with HBSS buffer with or without 100 or 300 ␮M LA, tied at both ends, and incubated at 37°C for
1 h. CCK (C) and secretin (D) release was determined by RIA or ELISA, respectively. Data are means ⫾ se (n⫽3 separate
mice per genotype per treatment). Release data are expressed as fold increase above buffer controls without LA. *P ⬍ 0.05;
**P ⬍ 0.01; ***P ⬍ 0.001.

observed in CD36⫺/⫺ segments with 100 ␮M LA, and a
1.6-fold enhancement occurred at 300 ␮M. Secretin
release from WT segments increased 3.3- and 4.3-fold
above basal in response to 100 and 300 ␮M LA. In
contrast, the same LA concentrations increased re-
lease only 1.4- and 1.8-fold in CD36⫺/⫺ segments.
These data suggested a major role of CD36 in
fat-induced EEC secretion of CCK and secretin that
was independent of the effects of CD36 on luminal
fat absorption.
CD36 expression in the small intestine and
colocalization with chromogranin A
CD36 is expressed apically on enterocytes of the small
intestine (39) after a proximal to distal decreasing
gradient (24, 25), and its levels are down-regulated by
small amounts of dietary fat (40). Information about
CD36 expression on EECs and whether it is regulated
by dietary fat is unavailable so this was examined.
Consistent with previous observations, CD36 was abun-
dant on epithelial cells of the duodenum and jejunum
and less abundant in the ileum (Supplemental Fig.
S1A⫺C). No CD36 signal was detected in intestines
from CD36⫺/⫺ mice (Supplemental Fig. S1D). Epithe-
lial CD36 expression was down-regulated in intestines
from fed (Supplemental Fig. S2C, D) compared with
unfed mice (Supplemental Fig. S2A, B). CD36 was
expressed on nonepithelial cells throughout the small
intestine, and nonepithelial expression also declined
with feeding (Supplemental Fig. S2).
We next examined expression of CD36 on EECs.
Chromogranin A is an essential component of secretory
vesicles typical of EECs and a marker for most cell types
of the enteroendocrine lineage (41). We costained for
chromogranin A and CD36 in intestinal sections and
found that a subset of chromogranin A-positive cells
express CD36 in the proximal (Fig. 2A) and distal small
intestine (data not shown), as determined by merge of
fluorescent signals. To quantify the distribution of
CD36 across chromogranin A-positive cells, doubly
positive cells (for chromogranin A and CD36) were
counted and expressed as a percentage of cells positive
for chromogranin A only. Sections from 5 mice were
used for quantification. This process demonstrated that
⬃35 and 16% of chromogranin A-positive EECs in the
proximal and distal small intestine, respectively, stained
for both CD36 and chromogranin A (Table 2). Images
at ⫻200 showed that CD36 is expressed on one-third of

1194 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org SUNDARESAN ET AL.
 Figure 2. CD36 expression on intestinal EECs.
A) CD36 (green) on chromogranin A (red)-positive
EECs. B, C) Colocalization of CD36 with secretin
(red)-positive cells. CD36 staining may be present on
apical membranes (arrows) of secretin-positive cells.
Nuclei are blue (DAPI). Under the same conditions, no CD36 signal is detected in CD36⫺/⫺ intestines (Supplemental Fig.
S1D). Immunoreactivity was visualized using Alexa Fluor 488 and 594 conjugates and a Zeiss fluorescence microscope
(⫻400). D, E) CD36 is expressed on one-third of chromogranin A-positive EECs (D) and on one-fifth of secretin-positive (E)
EECs (⫻200). Formalin-fixed, paraffin-embedded sections were deparaffinized and incubated sequentially with relevant
antibodies (Supplemental Table S1). Five unfed mice (overnight food withdrawal) were tested per peptide (n⫽5). Similar
results were obtained with fed mice (data not shown).

chromogranin A-positive cells (Fig. 2D) in the proximal
small intestine.
CD36 is expressed on subpopulations of EECs
Our in vivo data showed that CD36 deletion reduced
release of secretin and CCK. We examined whether
CD36 expression can be detected on the subpopula-
tions of EECs involved in release of these peptides. The
CD36 signal was detected in the cytoplasm of a subset of
secretin-producing cells in the proximal (Fig. 2B, C)
and distal (data not shown) small intestines. Approxi-
mately 21 and 9% of secretin-positive cells in the
proximal and distal small intestine, respectively, ex-
pressed CD36 (Table 2 and Fig. 2E). Secretin-positive
cells in these sections were also positive for chromo-
granin A (data not shown). EECs project their apical
membrane into the intestinal lumen, and all secretin-
positive EECs may express CD36 on their apical mem-
brane (Fig. 2B, C; arrows).
Colocalization of CD36 and CCK was studied using trans-
genic mice expressing the GFP driven by the CCK promoter.
A small subset of CCK-secreting cells (⬃5%) exhibited
intracellular costaining for CD36 (Fig. 3A, B and Table 2).
Based on immunofluorescence staining, CCK-positive cells
may have apical CD36 expression (Fig. 3C, D; arrows).
CD36 enhances secretin and CCK release by STC-1 cells

To demonstrate direct CD36 involvement in FA-in-

TABLE 2. Distribution of CD36 across EECs positive for chromogranin duced peptide secretion and to better understand the
A, secretin, and CCK along the duodenal-ileum axis in SI
signaling mechanisms involved, studies were conducted
Doubly positive cells
in the EEC line STC-1 derived from an endocrine
tumor of the mouse small intestine. The STC-1 cell line
Chromogranin A Secretin and is a mix of EEC subpopulations (42) that secrete a
Location and CD36 CD36 CCK and CD36
myriad of gut hormones including secretin and CCK
Proximal SI 28/80 (35.0%) 8/39 (20.5%) 5/100 (5%) (43) and has been used extensively to investigate the
Distal SI 4/25 (16.0%) 1/11 (9.1%) mechanisms underlying peptide release. No expression
of CD36 in STC-1 cells was detected, and cells stably
Sections from 5 individual mice were used for determination of expressing WT human CD36 and a control line express-
colocalization of CD36 with chromogranin A and secretin; sections ing empty vector were generated (Fig. 4A). A cell line
from 3 mice were used for CCK. Colocalization, determined by
doubly positive cells, is expressed as a percentage (n⫽5). SI, small stably expressing a mutated form of CD36 (CD36K/A)
intestine. that lacks CD36-mediated signaling to intracellular

CD36 MEDIATES RELEASE OF GUT PEPTIDES 1195

Figure 3. CD36 expression on CCK-positive cells
in proximal intestines of CCK-GFP transgenic
mice. A, B) Subset of CCK-positive cells (green)
express cytoplasmic CD36 (red). C, D) CD36 may
also be present on apical membranes (arrows) of
CCK-positive cells. Frozen OCT-embedded sec-
tions were fixed in 10% formalin, blocked, and
incubated with CD36 and GFP antibodies. Immu-
noreactivity was visualized as in the legend to Fig.
2 (⫻400). Three mice [fed (A, B) or unfed
overnight (C, D)] were tested for CCK (n⫽3).
calcium was also used. The C terminus of CD36 is
important for signal transduction (44, 45). In the
CD36K/A mutant lysines K469 and K472 in the C
terminus were substituted with alanine. As we reported
previously, the CD36K/A mutant has lost the ability to
regulate calcium influx, calcium-induced phospho-
lipase activation, and translocation to membranes (20),
but it has normal FA uptake activity (38). We hypothe-
sized that this mutant might have impaired regulation
of granule exocytosis as a result of its defect in calcium
signaling. All cells were treated with 50, 100, or 200 ␮M
FA (bound to 5 ␮M BSA), and the effect on peptide
secretion was monitored.
Secretin release was induced 2-fold above basal by 50
␮M DHA, in CD36-expressing STC-1 cells, and no
enhancement was observed in cells expressing either
CD36K/A or the empty vector (Fig. 4B). Similar data
were observed with LA (Fig. 4C) and linolenic acid
(data not shown). OA and PA were ineffective (Fig. 4D,
E). As the FA concentration was increased to 100 ␮M,
secretin release increased to 3.5 times basal with DHA
(Fig. 4B) and to 3 and 2 times basal with LA (Fig. 4C)
and OA (Fig. 4D), respectively. However, modest en-
hancement of release (1.5–1.8 times basal) could now
be observed in control (vector and CD36K/A) cells. At
200 ␮M FA, the stimulatory effects of DHA, LA, and OA
on secretin release were slightly more than those at 100
␮M, and PA had a small effect. To further validate the
CD36 specificity of observed effects, the irreversible
CD36 inhibitor sulfo-N-succinimidyl oleate (SSO; ref.
46) was used. In cells preincubated with SSO (20 ␮M,
15 min) and then treated with 100 ␮M FA, secretin
release was blunted (Fig. 4F), whereas no significant
effect was observed on CD36-independent release (vec-
tor and CD36K/A-expressing cells).
CCK
DHA at 50 ␮M stimulated CCK release by 2-fold from
cells expressing CD36 with no effect on empty vector or
CD36K/A-expressing cells (Fig. 5A). Similar effects
were observed with LA (Fig. 5B). PA induced a modest
30% increase (Fig. 5D), whereas OA was ineffective
(Fig. 5C). At higher concentrations of FA (200 ␮M),
1196 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org
DHA (Fig. 5A) and LA (Fig. 5B) induced almost 3-fold
enhancement of CCK release, whereas PA (Fig. 5D)
increased it by approximately 2-fold. OA was still inef-
fective. No enhancement was observed in cells express-
ing vector or CD36K/A. The specificity of CD36 in-
volvement was validated further by SSO producing
almost complete inhibition of CCK release (Fig. 5E).
Enterocyte coculture on secretin and CCK release by
STC-1 cells
Our experiments using intestinal segments in vitro
suggested that CD36 regulation of EEC release of
secretin and CCK was independent of changes in fat
absorption (Fig. 1). To examine this result further we
tested whether CD36 expression on enterocytes influ-
ences peptide secretion by neighboring EECs. Studies
were conducted using coculture of STC-1 cells with
Caco-2 cells, a well-studied model of enterocytes (47–
49). Differentiated Caco-2 cells with stable expression
of CD36, which are responsive to FA signaling (data not
shown), were generated and used for these experi-
ments. Cocultures of Caco-2 and STC-1 cells, with (⫹)
or without (⫺) stable expression of CD36 were tested
for LA-induced release of secretin and CCK. At 50 ␮M,
LA enhanced secretin release in STC-1 cells stably
expressing CD36 (⫹), compared with that in empty
vector controls (⫺) by⬃2-fold above basal whether the
coculture contained Caco-2 cells expressing CD36 (⫹)
or empty vector (⫺) (Fig. 4G). A similar trend was
observed with 200 ␮M LA. Secretin release increased
similarly and ⬃4-fold over basal in cocultures contain-
ing STC-1 (⫹) cells whether the cocultured Caco-2 cells
did or did not express CD36. In contrast with 50 ␮M
LA, 200 ␮M LA enhanced secretion ⬃2-fold in STC-1
cells not expressing CD36 (Fig. 4G). For CCK release,
the trends were similar to those observed with secretin
(Fig. 5F). LA at 50 and 200 ␮M stimulated CCK release
by 2- to 2.2- and 2.7- to 2.9-fold, respectively, in STC-1
cells stably expressing CD36 (⫹) compared with that in
vector controls (⫺), independent of CD36 expression
in Caco-2 cells. These data suggested limited contribu-
tion of CD36 expression on adjacent enterocytes to
FA-stimulated peptide release.
SUNDARESAN ET AL.
Figure 4. CD36 enhances FA-induced secretin release from STC-1 cells. A) Western blot showing CD36 expression in STC-1 cells
stably expressing empty vector, human CD36 WT, or the CD36K/A mutant. Ran is the loading control. B⫺E) Secretin release.
STC-1 cells were incubated with 50, 100, and 200 ␮M FA (with 5 ␮M BSA) or BSA alone (control) in HBSS (no glucose) for 60
min at 37°C, and secretin release was measured after DHA (B), LA (C), OA (D), and PA (E). Release is expressed as fold increase
over BSA controls after normalization to cell protein. Data are means ⫾ se of triplicates from 4 experiments (n⫽12).
F) LA-induced secretin release in the presence of the CD36 inhibitor, SSO (20 ␮M, 15 min). Cells were preincubated with 20
␮M SSO for 15 min before stimulation with 100 ␮M LA. G) Coculture of Caco-2 and STC-1 cells, with or without stable expression
of CD36. STC-1 cells expressing CD36 (STC ⫹) or empty vector (STC ⫺) were seeded with Caco-2 cells with (Caco ⫹) or without
(Caco ⫺) CD36 expression. Cocultures were serum starved overnight and then were incubated in HBSS with 50 or 200 ␮M LA
(plus 5 ␮M BSA) or BSA alone (controls) for 60 min at 37°C. Secretin release was determined and is expressed as fold increase
over BSA alone after normalization to cell protein. Data are means ⫾ se of triplicates from 3 experiments (n⫽8 –12). Points with
different letter symbols are significantly different (P⬍0.05). **P ⬍ 0.01.

CD36 MEDIATES RELEASE OF GUT PEPTIDES 1197

Figure 5. CD36 mediates FA-induced CCK release. A⫺D) CCK release from STC-1 cells expressing empty vector, CD36 WT, or
the mutant CD36K/A in response to DHA (A), LA (B), OA (C), and PA (D). E) LA-induced CCK release after treatment with
SSO (20 ␮M, 15 min). Cells were preincubated with 20 ␮M SSO for 15 min before stimulation with 100 ␮M LA. STC-1 cells were
FA treated (as in the legend to Fig. 4), and CCK release was determined by RIA. Data are means ⫾ se of triplicates from 3
experiments (n⫽9). Data points with different letter symbols are significantly different (P⬍0.05). *P ⬍ 0.05; **P ⬍ 0.01.
F) Coculture of Caco-2 and STC-1 cells, with (⫹) or without (⫺) stable expression of CD36. Cocultures were serum starved for
8 h and then were tested for CCK release as in the legend to Fig. 4. CCK was determined by RIA and is expressed as fold over
cells with BSA alone after normalization to cell protein. Data are means ⫾ se of triplicates from 2 experiments (n⫽4 for HBSS
controls; n⫽6 for treatments). Points with different letter symbols are significantly different (P⬍0.05). **P ⬍ 0.01.

cAMP production is involved in CD36-dependent
peptide release
cAMP regulates a variety of secretory events including
EEC release of secretin and CCK (30, 50). Its role in
CD36-mediated effects was examined. LA treatment
increased intracellular cAMP (⬃50%) compared with
that in BSA controls, and this effect was strongly
amplified in CD36-expressing cells in which cAMP
increased by 240% (Fig. 6A). DHA increased cAMP
(⬃88%) only in these cells, whereas PA had no effect
(Fig. 6A). Further, the cAMP inhibitor MDL12230A (10
␮M, 30 min) reduced CD36-dependent release of both
secretin (Fig. 6B) and CCK (Fig. 6C). MDL12230A had
no significant effect on release of these peptides in the
CD36K/A mutant. These data implicated cAMP gener-
ation in CD36-mediated FA-induced release of secretin
and CCK.
CD36-mediated secretin release is dependent on PKA
activation
A major effector of cAMP is PKA, which, when acti-
vated, phosphorylates an array of protein substrates
(51). We determined the effect of FA on PKA activity by
measuring phosphorylation of its substrates (RRXS*/T*
motifs). Addition of LA (Fig. 7A) or DHA (data not
shown) in the presence of 0.5 mM IBMX (to block

Figure 6. CD36-mediated enhancement of peptide release involves cAMP generation. A) Intracellular cAMP levels in response
to LA, DHA, and PA in STC-1 cells with vector, CD36 WT, and CD36K/A. STC-1 cells were FA treated at the indicated
concentration for 30 min (in the presence of the phosphodiesterase inhibitor IBMX, 0.5 mM), and cAMP was measured in cell
lysates. B, C) Secretin (B) and CCK (C) release after treatment with the cAMP inhibitor MDL12230A (10 ␮M, 30 min) in the
presence of 100 ␮M LA. Data are means ⫾ se of triplicates from 3 experiments (n⫽9). **P ⬍ 0.01; ***P ⬍ 0.001.

1198 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org SUNDARESAN ET AL.
Figure 7. CD36-mediated effects on peptide release involve activation of PKA and CaM-KII. A, B) Phospho-PKA substrates (with
RRXS*/T* motif) in cell lysates (A) and PKA C-␣ in nuclear fractions (B) of STC-1 cells expressing vector, CD36 WT, or
CD36K/A after LA stimulation (in the presence of 0.5 mM IBMX). Histone H-3 was used as a loading control for nuclear lysates
(n⫽4). C, D) LA-induced secretin (C) and CCK release (D) after treatment with the PKA inhibitor H-89. E) Representative
intracellular calcium transients in STC-1 cells stably expressing vector, CD36 WT, or CD36K/A. F) LA (100 ␮M) induced CCK
release in CD36-expressing STC-1 cells with or without treatment with the CaM-KII inhibitor KN-62 (2.5 ␮M, 15 min). Data are
means ⫾ se of triplicates from 3 experiments (n⫽9). *P ⬍ 0.05; **P ⬍ 0.01.

cAMP degradation) strongly enhanced phosphoryla-
tion of PKA substrates in STC-1 cells expressing WT
CD36 but not in cells expressing the signaling impaired
mutant CD36K/A (Fig. 7A). Under the same condi-
tions, PA treatment was ineffective (Supplemental Fig.
S3). Activation of the PKA tetramer associates with
translocation of the catalytic subunit to the nucleus
(51). LA treatment induced nuclear translocation of
catalytic PKA C-␣ in control cells, and this effect was
amplified 3-fold in cells expressing CD36 WT (Fig. 7B).
LA increased nuclear protein kinase C content by
⬃2.4-fold in vector cells, by 7.4-fold in CD36WT cells,
and by 1.7-fold in CD36K/A cells. The responses of
vector and CD36K/A-expressing cells were similar and
significantly less than that of CD36WT cells (P⬍0.01).
Pretreatment with the PKA inhibitor H-89 (10 ␮M, 30
min) reduced secretin release only in CD36 WT-ex-
pressing cells (Fig. 7C), suggesting that release involved
PKA activation. In contrast, CD36-mediated, FA-in-
duced CCK release was not affected by H-89 (Fig. 7D),
implicating PKA-independent mechanisms downstream of
cAMP.
CD36-mediated CCK release involves CaM-KII
An increase in intracellular calcium after cAMP gener-
ation has been implicated in release of CCK (50, 52).
CD36 was recently reported to influence calcium flux
in Chinese hamster ovary, macrophages, and taste bud
cells (20, 22). We investigated effect of CD36 expres-
sion on calcium transients in response to LA in the
absence or presence of extracellular calcium. A rapid
increase in intracellular calcium was observed in LA-
treated STC-1 cells expressing CD36 WT on addition of
medium calcium, whereas no effect was observed in
cells expressing the vector or CD36K/A mutant (Fig.
7E). Calcium influx activates CaM-KII, which is involved
in release of CCK (32). Indeed, treatment with the
CaM-KII inhibitor KN-62 (2.5 ␮M, 15 min) blunted
CCK release in CD36-expressing cells (Fig. 7F), sup-
porting involvement of CaM-KII in CD36-mediated
CCK release.
DISCUSSION
CD36 contributes to different steps of the fat absorp-
tion process (53); it mediates fat perception in taste
bud cells and the initiation of the cephalic phase of
digestion (22). It also influences intestinal fat process-
ing and chylomicron formation (25, 26, 28). This study
documented a novel and potentially important meta-
bolic function of CD36: its mediation of gut peptide
release in response to dietary FA. We showed that CD36
is expressed on EECs positive for secretin and CCK and
the release of these peptides in response to intragastric

CD36 MEDIATES RELEASE OF GUT PEPTIDES 1199

fat was halved in CD36⫺/⫺ mice. This reduction was
most likely independent of changes in fat absorption
because it was reproduced in isolated intestinal seg-
ments. Using the EEC line STC-1 with stable expres-
sion of CD36 or a signaling-impaired CD36 mutant,
specific CD36 mediation of FA-induced peptide re-
lease was demonstrated to involve increases in cellu-
lar cAMP and calcium.
CD36 regulates peptide release
CD36 deletion was associated with 50 – 60% lower
plasma levels of secretin and CCK at 30 min after an
intragastric triglyceride load. In vitro, the effect of FA to
induce release of these peptides was specific to cells
expressing functional WT CD36. The calcium signal-
ing-impaired CD36K/A mutant was ineffective despite
the fact that it expresses normally, localizes to the
plasma membrane, and functions in FA uptake (20,
38). Our findings using WT and CD36⫺/⫺ mice, given
a high oil load, support the existence of both CD36-
dependent and CD36-independent peptide release be-
cause secretin and CCK release was suppressed by 60
and 50%, respectively. The work in vitro with proximal
intestinal segments and with STC-1 cells showed that
the CD36-independent component becomes more ap-
parent as the FA concentration is increased and the
high-affinity CD36-mediated pathway is saturated. In
vivo, this could imply that one function of CD36 is to
enable secretin and CCK release at relatively low FA
concentrations possibly early during the digestion pro-
cess, thus optimizing subsequent absorption. Other FA
receptors such as GPR120 (17) and GPR40 (2), which
are localized more distally in the intestine, would
contribute to FA-induced FA release at a later stage in
the absorptive process. GPR120 has been mainly impli-
cated in mediation of FA-induced incretin release (17,
18). Its role in CCK release has been briefly docu-
mented in STC-1 cells, in which small interfering RNA
directed against GPR120 but not GPR40 inhibited a
2-fold effect of linolenic acid to induce CCK secretion
by 30 – 40% (54). More convincing evidence docu-
mented involvement of GPR40 in long-chain FA-in-
duced release of CCK (2) in vivo using GPR40⫺/⫺ mice.
A load of intragastric olive oil comparable to that used
in our study resulted at 30 min in a 4.2-fold increase of
plasma CCK vs. a 2.8-fold increase in GPR40⫺/⫺ mice,
meaning a reduction of 33%. These findings together
with our observation of a 50% reduction of plasma
CCK, 30 min after oil gavage in CD36⫺/⫺ mice (Fig. 1)
support the interpretation that CD36 and GPR40 might
account for most FA-induced CCK release in vivo. In
the case of secretin, our study provides, to our knowl-
edge, the first information related to the FA receptor
involved in secretin release by documenting a major
role of CD36 based on the 60% reduction of plasma
secretin release in CD36⫺/⫺ mice, given a high intra-
gastric oil load (Fig. 1).
1200 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org
CD36-mediated FA signaling enhances cAMP
generation and calcium transients
Endocrine cells release granules in response to factors
that increase intracellular Ca2⫹ or cAMP levels. The
cAMP pathway modulates granule exocytosis in a vari-
ety of cells (51) and has been implicated in release of
gut peptides (30, 50). We showed that CD36 signaling
regulates cAMP levels and that this was involved in its
enhancement of FA-induced release of secretin and
CCK. FA activation of PKA, the major cAMP effector,
and enhanced nuclear PKA content were documented
in CD36-expressing cells. However, only release of
secretin was markedly attenuated by the PKA inhibitor
H-89, whereas that of CCK was not, suggesting PKA-
independent mechanisms. CCK release is triggered by
an increase in intracellular Ca2⫹ that is coupled to
cAMP generation (50) and CaM-KII activation (32).
Consistent with CD36 regulation of this pathway, FA
induced an increase in intracellular Ca2⫹ specifically in
STC-1 cells expressing CD36, and the CCK response
was suppressed by the CaM-KII inhibitor KN-62. The
differential effects of signaling inhibitors, implying
involvement of distinct pathways downstream of cAMP
in the regulation of secretin vs. CCK release, probably
reflect the contribution of different cell subpopulations
because the STC-1 cell line derived from an endocrine
tumor of the intestine is heterogeneous (42, 43). Our
data with STC-1 cells are consistent with previous work
supporting involvement of Ca2⫹ and cAMP in release of
secretory granules in most cell types and with selectivity
of granule exocytosis, reflecting variable sensitivity to
Ca2⫹ and selective downstream phosphorylation path-
ways (55). Some EECs in the mouse intestine synthesize
and secrete more than one peptide, including CCK and
secretin (56). There is also evidence for different
composition of granules in CCK- or secretin-releasing
EECs (57), consistent with differential regulation of
granule release. The possibility that some secretion of
CCK and secretin may occur from the same STC-1
subpopulation cannot be ruled out.
In summary, our data document CD36 expression on
secretin- and CCK-releasing EECs and its role in FA-
induced release of these peptides in vivo. The more
abundant localization of CD36 in the proximal than in
the distal intestine and the relatively higher FA affinity
of CD36-mediated vs. CD36-independent peptide re-
lease suggest that CD36 would play a critical role in the
early phase of fat absorption. Other FA receptors such
as GPR40, which is more abundant in the distal intes-
tine, would contribute at later phases of absorption.
Our data, together with previous findings, suggest that
CD36 and GPR40 account for most FA-induced CCK
release and that CD36 is the predominant regulator of
FA-induced secretin release. CCK and secretin play
important regulatory roles in fat absorption because
they influence gastric emptying and acid secretion and
pancreatic and intestinal bicarbonate secretion in ad-
dition to gallbladder contractility and intestinal motility
(3, 58). Both peptides influence satiety and energy
SUNDARESAN ET AL.
homeostasis (12, 13). Regulation of FA-induced CCK
and secretin release contributes to the role of CD36 in
intestinal handling of dietary fat. In humans, common
SNPs in the CD36 gene influence plasma lipids (59),
and CD36 deficiency is characterized by altered chylo-
micron production (60). In addition, SNPs that reduce
the CD36 level associate with diminished oral FA sen-
sitivity (23). Whether intestinal FA sensing is also
altered in these subjects remains to be determined. FA
sensing in the small intestine with consequent effects
on gut peptide release and on satiety were reported to
be attenuated in subjects with reduced oral FA sensitiv-
ity or after high-fat feeding (8).
The authors acknowledge valuable assistance by Terri
Pietka (Adipocyte Biology and Molecular Nutrition Core,
Nutrition and Obesity Research Center, Washington Univer-
sity School of Medicine; P30-DK056341) and are grateful to
Dr. Ondrej Kuda for help with the calcium flux studies and to
Dr. Doug Hanahan for the permission to obtain STC-1 cells
from American Type Culture Collection (Manassas, VA,
USA). This work was supported by funding from U.S. Na-
tional Institutes of Health grants DK033301, DK60022 (to
N.A.A.), DK33165, DK55753 (to W.F.S.), and DK091946 (to
R.A.L.). R.S., T.E.R., R.C., and F.N. assisted in data acquisition
and reviewed the manuscript; W.F.S. and R.A.L. critically
reviewed the manuscript; S.S. obtained and analyzed data;
and S.S. and N.A.A. were involved in development of the
study concept and design, data analysis and interpretation,
and manuscript preparation.
REFERENCES
1. Beglinger, C., and Degen, L. (2004) Fat in the intestine as a
regulator of appetite—role of CCK. Physiol. Behav. 83, 617–621
2. Liou, A. P., Lu, X., Sei, Y., Zhao, X., Pechhold, S., Carrero, R. J.,
Raybould, H. E., and Wank, S. (2011) The G-protein-coupled
receptor GPR40 directly mediates long-chain fatty acid-induced
secretion of cholecystokinin. Gastroenterology 140, 903–912
3. Liddle, R. A. (2000) Regulation of cholecystokinin secretion in
humans. J. Gastroenterol. 35, 181–187
4. Buchan, A. M., Polak, J. M., Solcia, E., Capella, C., Hudson, D.,
and Pearse, A. G. (1978) Electron immunohistochemical evi-
dence for the human intestinal I cell as the source of CCK. Gut
19, 403–407
5. Lam, I. P., Lee, L. T., Choi, H. S., and Chow, B. K. (2006)
Localization of small heterodimer partner (SHP) and secretin
in mouse duodenal cells. Ann. N. Y. Acad. Sci. 1070, 371–375
6. Polak, J. M., Bloom, S., Coulling, I., and Pearse, A. G. (1971)
Immunofluorescent localization of secretin in the canine duo-
denum. Gut 12, 605–610
7. Polak, J. M., Coulling, I., Bloom, S., and Pearse, A. G. (1971)
Immunofluorescent localization of secretin and enteroglucagon
in human intestinal mucosa. Scand. J. Gastroenterol. 6, 739 –744
8. Little, T. J., and Feinle-Bisset, C. (2011) Effects of dietary fat on
appetite and energy intake in health and obesity— oral and
gastrointestinal sensory contributions. Physiol. Behav. 104, 613–
620
9. Sclafani, A., and Ackroff, K. (2012) Role of gut nutrient sensing
in stimulating appetite and conditioning food preferences. Am.
J. Physiol. Regul. Integr. Comp. Physiol. 302, R1119 –R1133
10. Berna, M. J., and Jensen, R. T. (2007) Role of CCK/gastrin
receptors in gastrointestinal/metabolic diseases and results of
human studies using gastrin/CCK receptor agonists/antago-
nists in these diseases. Curr. Top. Med. Chem. 7, 1211–1231
11. Chey, W. Y., and Chang, T. M. (2003) Secretin, 100 years later.
J. Gastroenterol. 38, 1025–1035
12. Cheng, C. Y., Chu, J. Y., and Chow, B. K. (2011) Central and
peripheral administration of secretin inhibits food intake in
CD36 MEDIATES RELEASE OF GUT PEPTIDES
mice through the activation of the melanocortin system. Neuro-
psychopharmacology 36, 459 –471
13. Dockray, G. J. (2012) Cholecystokinin. Curr. Opin. Endocrinol.
Diabetes Obes. 19, 8 –12
14. Hajri, T., and Abumrad, N. A. (2002) Fatty acid transport across
membranes: relevance to nutrition and metabolic pathology.
Annu. Rev. Nutr. 22, 383–415
15. Oh, D. Y., and Lagakos, W. S. (2011) The role of G-protein-
coupled receptors in mediating the effect of fatty acids on
inflammation and insulin sensitivity. Curr. Opin. Clin. Nutr.
Metab. Care 14, 322–327
16. Vinolo, M. A., Hirabara, S. M., and Curi, R. (2012) G-protein-
coupled receptors as fat sensors. Curr. Opin. Clin. Nutr. Metab.
Care 15, 112–116
17. Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T.,
Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G.
(2005) Free fatty acids regulate gut incretin glucagon-like
peptide-1 secretion through GPR120. Nat. Med. 11, 90 –94
18. Oh, D. Y., Talukdar, S., Bae, E. J., Imamura, T., Morinaga, H.,
Fan, W., Li, P., Lu, W. J., Watkins, S. M., and Olefsky, J. M.
(2010) GPR120 is an omega-3 fatty acid receptor mediating
potent anti-inflammatory and insulin-sensitizing effects. Cell
142, 687–698
19. El-Yassimi, A., Hichami, A., Besnard, P., and Khan, N. A. (2008)
Linoleic acid induces calcium signaling, Src kinase phosphory-
lation, and neurotransmitter release in mouse CD36-positive
gustatory cells. J. Biol. Chem. 283, 12949 –12959
20. Kuda, O., Jenkins, C. M., Skinner, J. R., Moon, S. H., Su, X.,
Gross, R. W., and Abumrad, N. A. (2011) CD36 protein is
involved in store-operated calcium flux, phospholipase A2 acti-
vation, and production of prostaglandin E2. J. Biol. Chem. 286,
17785–17795
21. Khan, N. A., and Besnard, P. (2009) Oro-sensory perception of
dietary lipids: new insights into the fat taste transduction.
Biochim. Biophys. Acta 1791, 149 –155
22. Laugerette, F., Passilly-Degrace, P., Patris, B., Niot, I., Febbraio,
M., Montmayeur, J. P., and Besnard, P. (2005) CD36 involve-
ment in orosensory detection of dietary lipids, spontaneous fat
preference, and digestive secretions. J. Clin. Invest. 115, 3177–
3184
23. Pepino, M. Y., Love-Gregory, L., Klein, S., and Abumrad, N. A.
(2012) The fatty acid translocase gene CD36 and lingual lipase
influence oral sensitivity to fat in obese subjects. J. Lipid Res. 53,
561–566
24. Lobo, M. V., Huerta, L., Ruiz-Velasco, N., Teixeiro, E., de la
Cueva, P., Celdran, A., Martin-Hidalgo, A., Vega, M. A., and
Bragado, R. (2001) Localization of the lipid receptors CD36 and
CLA-1/SR-BI in the human gastrointestinal tract: towards the
identification of receptors mediating the intestinal absorption
of dietary lipids. J. Histochem. Cytochem. 49, 1253–1260
25. Nassir, F., Wilson, B., Han, X., Gross, R. W., and Abumrad, N. A.
(2007) CD36 is important for fatty acid and cholesterol uptake
by the proximal but not distal intestine. J. Biol. Chem. 282,
19493–19501
26. Drover, V. A., Ajmal, M., Nassir, F., Davidson, N. O., Nauli,
A. M., Sahoo, D., Tso, P., and Abumrad, N. A. (2005) CD36
deficiency impairs intestinal lipid secretion and clearance of
chylomicrons from the blood. J. Clin. Invest. 115, 1290 –1297
27. Hsieh, J., Longuet, C., Maida, A., Bahrami, J., Xu, E., Baker,
C. L., Brubaker, P. L., Drucker, D. J., and Adeli, K. (2009)
Glucagon-like peptide-2 increases intestinal lipid absorption
and chylomicron production via CD36. Gastroenterology 137,
997–1005, 1005.e1⫺1005.e4
28. Nauli, A. M., Nassir, F., Zheng, S., Yang, Q., Lo, C. M.,
Vonlehmden, S. B., Lee, D., Jandacek, R. J., Abumrad, N. A.,
and Tso, P. (2006) CD36 is important for chylomicron forma-
tion and secretion and may mediate cholesterol uptake in the
proximal intestine. Gastroenterology 131, 1197–1207
29. Edfalk, S., Steneberg, P., and Edlund, H. (2008) Gpr40 is
expressed in enteroendocrine cells and mediates free fatty acid
stimulation of incretin secretion. Diabetes 57, 2280 –2287
30. Chang, C. H., Chey, W. Y., Erway, B., Coy, D. H., and Chang,
T. M. (1998) Modulation of secretin release by neuropeptides in
secretin-producing cells. Am. J. Physiol. 275, G192–G202
31. Parker, H. E., Reimann, F., and Gribble, F. M. (2010) Molecular
mechanisms underlying nutrient-stimulated incretin secretion.
Expert Rev. Mol. Med. 12, e1
1201
32. Prpic, V., Basavappa, S., Liddle, R. A., and Mangel, A. W. (1994)
Regulation of cholecystokinin secretion by calcium-dependent
calmodulin kinase II: differential effects of phenylalanine and
cAMP. Biochem. Biophys. Res. Commun. 201, 1483–1489
33. Chandra, R., Samsa, L. A., Vigna, S. R., and Liddle, R. A. (2010)
Pseudopod-like basal cell processes in intestinal cholecystokinin
cells. Cell Tissue Res. 341, 289 –297
34. Suzuki, S., Suzuki, H., Horiguchi, K., Tsugawa, H., Matsuzaki, J.,
Takagi, T., Shimojima, N., and Hibi, T. (2010) Delayed gastric
emptying and disruption of the interstitial cells of Cajal network
after gastric ischaemia and reperfusion. Neurogastroenterol. Motil.
22, 585–593, e126
35. Liddle, R. A., Goldfine, I. D., and Williams, J. A. (1984) Bioassay
of plasma cholecystokinin in rats: effects of food, trypsin inhib-
itor, and alcohol. Gastroenterology 87, 542–549
36. Tashiro, M., Samuelson, L. C., Liddle, R. A., and Williams, J. A.
(2004) Calcineurin mediates pancreatic growth in protease
inhibitor-treated mice. Am. J. Physiol. Gastrointest. Liver Physiol.
286, G784 –G790
37. Rehfeld, J. F. (1998) Accurate measurement of cholecystokinin
in plasma. Clin. Chem. 44, 991–1001
38. Smith, J., Su, X., El-Maghrabi, R., Stahl, P. D., and Abumrad,
N. A. (2008) Opposite regulation of CD36 ubiquitination by
fatty acids and insulin: effects on fatty acid uptake. J. Biol. Chem.
283, 13578 –13585
39. Poirier, H., Degrace, P., Niot, I., Bernard, A., and Besnard, P.
(1996) Localization and regulation of the putative membrane
fatty-acid transporter (FAT) in the small intestine. Comparison
with fatty acid-binding proteins (FABP). Eur. J. Biochem. 238,
368 –373
40. Tran, T. T., Poirier, H., Clement, L., Nassir, F., Pelsers, M. M.,
Petit, V., Degrace, P., Monnot, M. C., Glatz, J. F., Abumrad,
N. A., Besnard, P., and Niot, I. (2011) Luminal lipid regulates
CD36 levels and downstream signaling to stimulate chylomicron
synthesis. J. Biol. Chem. 286, 25201–25210
41. Varndell, I. M., Lloyd, R. V., Wilson, B. S., and Polak, J. M.
(1985) Ultrastructural localization of chromogranin: a potential
marker for the electron microscopical recognition of endocrine
cell secretory granules. Histochem. J. 17, 981–992
42. Cheung, A. T., Dayanandan, B., Lewis, J. T., Korbutt, G. S.,
Rajotte, R. V., Bryer-Ash, M., Boylan, M. O., Wolfe, M. M., and
Kieffer, T. J. (2000) Glucose-dependent insulin release from
genetically engineered K cells. Science 290, 1959 –1962
43. Rindi, G., Grant, S. G., Yiangou, Y., Ghatei, M. A., Bloom, S. R.,
Bautch, V. L., Solcia, E., and Polak, J. M. (1990) Development of
neuroendocrine tumors in the gastrointestinal tract of trans-
genic mice. Heterogeneity of hormone expression. Am. J. Pathol.
136, 1349 –1363
44. Lipsky, R. H., Eckert, D. M., Tang, Y., and Ockenhouse, C. F.
(1997) The carboxyl-terminal cytoplasmic domain of CD36 is
required for oxidized low-density lipoprotein modulation of
NF-␬B activity by tumor necrosis factor-␣. Recept. Signal Trans-
duct. 7, 1–11
45. Silverstein, R. L. (2009) Type 2 scavenger receptor CD36 in
platelet activation: the role of hyperlipemia and oxidative stress.
Clin. Lipidol. 4, 767
46. Harmon, C. M., Luce, P., Beth, A. H., and Abumrad, N. A.
(1991) Labeling of adipocyte membranes by sulfo-N-succinimi-
1202 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org
dyl derivatives of long-chain fatty acids: inhibition of fatty acid
transport. J. Membr. Biol. 121, 261–268
47. Hussain, M. M. (2000) A proposed model for the assembly of
chylomicrons. Atherosclerosis 148, 1–15
48. Levy, E., and Bendayan, M. (2000) Use of immunoelectron
microscopy and intestinal models to explore the elaboration of
apolipoproteins required for intraenterocyte lipid transport.
Microsc. Res. Tech. 49, 374 –382
49. Mathias, A., Duc, M., Favre, L., Benyacoub, J., Blum, S., and
Corthesy, B. (2010) Potentiation of polarized intestinal Caco-2
cell responsiveness to probiotics complexed with secretory IgA.
J. Biol. Chem. 285, 33906 –33913
50. Scott, L., Prpic, V., Capel, W. D., Basavappa, S., Mangel, A. W.,
Gettys, T. W., and Liddle, R. A. (1996) ␤-Adrenergic regulation
of cholecystokinin secretion in STC-1 cells. Am. J. Physiol. 270,
G291–G297
51. Seino, S., and Shibasaki, T. (2005) PKA-dependent and PKA-
independent pathways for cAMP-regulated exocytosis. Physiol.
Rev. 85, 1303–1342
52. Sidhu, S. S., Thompson, D. G., Warhurst, G., Case, R. M., and
Benson, R. S. (2000) Fatty acid-induced cholecystokinin secre-
tion and changes in intracellular Ca2⫹ in two enteroendocrine
cell lines, STC-1 and GLUTag. J. Physiol. 528 (Pt. 1), 165–176
53. Abumrad, N. A., and Davidson, N. O. (2012) Role of the gut in
lipid homeostasis. Physiol. Rev. 92, 1061–1085
54. Tanaka, T., Katsuma, S., Adachi, T., Koshimizu, T. A., Hirasawa,
A., and Tsujimoto, G. (2008) Free fatty acids induce cholecysto-
kinin secretion through GPR120. Naunyn-Schmiedebergs Arch.
Pharmacol. 377, 523–527
55. Burgoyne, R. D., and Morgan, A. (2003) Secretory granule
exocytosis. Physiol. Rev. 83, 581–632
56. Habib, A. M., Richards, P., Cairns, L. S., Rogers, G. J., Bannon,
C. A., Parker, H. E., Morley, T. C., Yeo, G. S., Reimann, F., and
Gribble, F. M. (2012) Overlap of endocrine hormone expres-
sion in the mouse intestine revealed by transcriptional profiling
and flow cytometry. Endocrinology 153, 3054 –3065
57. Portela-Gomes, G. M., Stridsberg, M., Johansson, H., and Grime-
lius, L. (1997) Complex co-localization of chromogranins and
neurohormones in the human gastrointestinal tract. J. His-
tochem. Cytochem. 45, 815–822
58. Lam, I. P., Siu, F. K., Chu, J. Y., and Chow, B. K. (2008) Multiple
actions of secretin in the human body. Int. Rev. Cytol. 265,
159 –190
59. Love-Gregory, L., Sherva, R., Schappe, T., Qi, J. S., McCrea, J.,
Klein, S., Connelly, M. A., and Abumrad, N. A. (2011) Common
CD36 SNPs reduce protein expression and may contribute to a
protective atherogenic profile. Hum. Mol. Genet. 20, 193–201
60. Masuda, D., Hirano, K., Oku, H., Sandoval, J. C., Kawase, R.,
Yuasa-Kawase, M., Yamashita, Y., Takada, M., Tsubakio-
Yamamoto, K., Tochino, Y., Koseki, M., Matsuura, F., Nishida,
M., Kawamoto, T., Ishigami, M., Hori, M., Shimomura, I., and
Yamashita, S. (2009) Chylomicron remnants are increased in
the postprandial state in CD36 deficiency. J. Lipid Res. 50,
999 –1011
Received for publication July 30, 2012.
Accepted for publication November 26, 2012.
SUNDARESAN ET AL.
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