ABSTRACT Genetic variants in the fatty acid (FA) secretin. These peptides contribute to the role of CD36 in
translocase FAT/CD36 associate with abnormal post- fat absorption and to its pleiotropic metabolic effects.—
prandial lipids and influence risk for the metabolic Sundaresan, S., Shahid, R., Riehl, T. E., Chandra, R.,
syndrome. CD36 is abundant on apical enterocyte Nassir, F., Stenson, W. F., Liddle, R. A., Abumrad, N. A.
membranes in the proximal small intestine, where it CD36-dependent signaling mediates fatty acid-induced
facilitates FA uptake and FA-initiated signaling. We gut release of secretin and cholecystokinin. FASEB J. 27,
explored whether CD36 signaling influences FA-medi- 1191–1202 (2013). www.fasebj.org
ated secretion of cholecystokinin (CCK) and secretin,
peptides released by enteroendocrine cells (EECs) in
the duodenum/jejunum, which regulate events impor- Key Words: CCK 䡠 cAMP 䡠 calcium
tant for fat digestion and homeostasis. CD36 was
immunodetected on apical membranes of secretin- and Dietary fatty acids (FAs) trigger release of a number
CCK-positive EECs and colocalized with cytosolic gran- of peptides, including cholecystokinin (CCK) and se-
ules. Intragastric lipid administration to CD36ⴚ/ⴚ mice cretin, by enteroendocrine cells (EECs) (1, 2). CCK is
released less secretin (ⴚ60%) and CCK (ⴚ50%) com- secreted by I cells (3, 4) and secretin by S cells (5–7),
pared with wild-type mice. Likewise, diminished secre- localized primarily to the duodenum and jejunum.
tin and CCK responses to FA were observed with Both peptides influence fat absorption and participate
CD36ⴚ/ⴚ intestinal segments in vitro, arguing against in nutrient sensing and regulation of energy balance
influence of alterations in fat absorption. Signaling (1, 8, 9). CCK regulates gallbladder contraction, secre-
mechanisms underlying peptide release were examined tions by the stomach and acinar pancreas, and colonic
in STC-1 cells stably expressing human CD36 or a motility (10). Secretin plays an important role in buff-
signaling-impaired mutant (CD36K/A). FA stimulation ering the acidic chyme in the intestinal lumen by
of cells expressing CD36 (vs. vector or CD36K/A) stimulating water and bicarbonate secretion and en-
released more secretin (3.5- to 4-fold) and CCK (2- to hances CCK effects on the acinar pancreas and gall-
3-fold), generated more cAMP (2- to 2.5-fold), and bladder (11). In addition, both CCK and secretin have
enhanced protein kinase A activation. Protein kinase A prosatiety effects (12, 13).
inhibition (H-89) blunted secretin (80%) but not CCK A number of cellular surface receptors for FA, includ-
release, which was reduced (50%) by blocking of cal- ing the family of G-protein-coupled receptors (GPRs)
modulin kinase II (KN-62). Coculture of STC-1 cells and the scavenger receptor CD36, have been identified.
with Caco-2 cells stably expressing CD36 did not alter Long-chain FAs are recognized by GPR120, GPR40, and
secretin or CCK release, consistent with a minimal CD36 (14 –16). CD36 facilitates cellular FA uptake (14)
effect of adjacent enterocytes. In summary, CD36 is a and, like the GPRs (17, 18), mediates FA-induced signal
major mediator of FA-induced release of CCK and transduction, influencing cellular calcium for release of
neurotransmitters (19) and arachidonic acid and pros-
taglandins (20). In taste cells, FA sensing by CD36
Abbreviations: BSA, bovine serum albumin; CaM-KII, cal-
modulin kinase II; CCK, cholecystokinin; DHA, docosa- mediates fat perception and preference (21, 22) and
hexaenoic acid; DMEM, Dulbecco’s modified Eagle’s me- induces the cephalic phase of digestion (22). In hu-
dium; EEC, enteroendocrine cell; ELISA, enzyme-linked mans, oral FA detection thresholds are higher in sub-
immunosorbent assay; FA, fatty acid; FBS, fetal bovine serum;
GFP, green fluorescent protein; GPR, G-protein-coupled re-
1
ceptor; HBSS, Hanks’ balanced saline solution; IBMX, Correspondence: Department of Medicine, Center for
3-isobutyl-1-methylxanthine; LA, linoleic acid; OA, oleic acid; Human Nutrition, 660 S. Euclid Ave., Campus Box 8031, St.
PA, palmitic acid; PKA, protein kinase A; PMSF, phenylmeth- Louis, MO 63110, USA. E-mail: nabumrad@dom.wustl.edu
anesulfonyl fluoride; RIA, radioimmunoassay; SNP, single- doi: 10.1096/fj.12-217703
nucleotide polymorphism; SSO, sulfo-N-succinimidyl oleate; This article includes supplemental data. Please visit http://
WT, wild type www.fasebj.org to obtain this information.
CD36-null (CD36⫺/⫺) and WT mice on the C57BL/6 After overnight food withdrawal, mice were administered an
background were housed in a facility with a 12-h light-dark intragastric load of olive oil or saline (16 l/g body weight),
cycle and fed chow ad libitum (Purina, St. Louis, MO, USA). and peptide release into the blood was measured 30 min
Female mice, 3– 4 mo old, were denied access to food for later. CCK and secretin were extracted on C18 columns
16 h before euthanasia. Mouse care and use followed (Waters, Milford, MA, USA) and quantified. Plasma CCK was
guidelines of the animal ethics committee of Washington measured by bioassay using amylase secretion from isolated
University School of Medicine (St. Louis, MO, USA). rat pancreatic acini (35). Plasma from 3 mice was pooled to
Transgenic CCK-green fluorescent protein (GFP) mice, used obtain adequate sensitivity (36). Secretin was measured by
TABLE 1. Antibodies
1192 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org SUNDARESAN ET AL.
enzyme-linked immunosorbent assay (ELISA; Phoenix Phar- 37°C) in HBSS containing 1 mM 3-isobutyl-1-methylxanthine
maceuticals, Belmont, CA, USA). (IBMX). Cellular protein was determined in lysates from
duplicate wells (DC Protein Assay; Bio-Rad Laboratories,
Secretion by proximal intestinal loops 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␣.
The proximal small intestine isolated from WT and CD36⫺/⫺ Intracellular calcium was measured as described previously
mice after 16 h of food withdrawal was cut into two 10-cm (20), using Fura-2/AM dye (2.5 M).
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 Western blot analyses
phosphatase inhibitors), tied at both ends, and incubated for
1 h at 37°C. The media were collected, lyophilized, rehy- Protein signals were detected using the Odyssey Infrared
drated with 500 l, and assayed. Secretin was measured by System (Li-Cor Biosciences, Lincoln, NE, USA) as described
ELISA, and CCK was quantified by radioimmunoassay (RIA) previously (20). Cell proteins separated and transferred to
using 125I-labeled CCK-8 and antisera 92128, which recognize polyvinylidene fluoride membranes were blocked and incu-
biologically active CCK (37). bated overnight with primary antibodies (4°C) and then for 1
h with infrared dye-labeled secondary antibodies (room tem-
perature).
STC-1 and Caco-2 cells
Statistical analyses
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 Statistical analyses were performed with GraphPad Prism 4
expressing human WT or mutated CD36 (CD36K/A) with software (GraphPad Software Inc., San Diego, CA, USA).
C-terminal lysines 469 and 472 substituted by alanine (38) Differences obtained by 1-way ANOVA were considered sig-
were generated by electroporation (Nucleofector Kit V; nificant at values of P ⱕ 0.05. The Bonferroni test was
Lonza, Cologne, Germany) followed by selection in gentami- performed to identify groups that were different.
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. RESULTS
Cocultures of enterocytes (Caco-2 cells) with EECs (STC-1
cells) were established by serial seeding. First, 3.0 ⫻ 106
CD36⫺/⫺ mice show reduced levels of intestinal pep-
differentiated Caco-2 cells expressing CD36 or empty vector
were seeded in DMEM containing 10% FBS. Twelve hours tides in response to a lipid load: CD36 is abundant in
later, 1 ml of culture medium containing 0.3 ⫻ 106 STC-1 the proximal intestine, where it has been shown to
cells, with or without CD36 expression, was added to the dish. facilitate uptake of FA and cholesterol (25) and to
A 1:10 ratio of STC-1 cells to Caco-2 cells was chosen to promote chylomicron formation (26). We examined
simulate the relative physiological distribution. One day later, the influence of CD36 deletion on fat-induced secre-
the mixed culture was washed, serum starved for 8 h, and tion of CCK and secretin, peptides with important roles
incubated (60 min, 37°C) in HBSS (with Ca2⫹ and Mg2⫹)
in fat absorption that are released by EECs localized
with 50 and 200 M LA (plus 5 M BSA) or with BSA alone
(controls). Secretin and CCK release was determined by primarily in proximal segments (3–7). Plasma CCK and
ELISA and RIA (37), respectively. secretin levels were measured in WT and CD36⫺/⫺
mice 30 min after an intragastric load of olive oil.
Peptide release by STC-1 cells CD36⫺/⫺ mice had 50% lower CCK (Fig. 1A) and 60%
lower secretin levels (Fig. 1B) than WT mice. Peptide
Cells (1⫻106) in 6-well plates were serum starved in DMEM levels were similar in saline-administered WT and
and then were stimulated (1 h) by addition of 50 –200 M FA; CD36⫺/⫺ mice.
LA, docosahexaenoic acid (DHA), oleic acid (OA), and CD36 deficiency alters gut fat absorption by impair-
palmitic acid (PA) complexed to 5 M BSA were added in ing chylomicron formation and shifting more luminal
HBSS (no glucose) containing aprotinin (200 kallikrein- fat to distal parts of the small intestine (26, 28). These
inhibiting units/ml). Media were collected for secretin anal-
yses by ELISA. Cells were lysed for protein assays in buffer [20
changes might contribute to the reduction of fat-
mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM Na2EDTA; 1 mM induced CCK and secretin release in CD36⫺/⫺ mice by
EGTA; 1% Triton X-100; 2.5 mM sodium pyrophosphate; 1 altering fat exposure of EECs in the proximal part of
mM glycerol 2-phosphate; 1 mM Na3VO4; 1 g/ml leupeptin; the intestine. We first compared gastric emptying rates
and 1 mM phenylmethanesulfonyl fluoride (PMSF)] contain- in WT and CD36⫺/⫺ mice and found no significant
ing a protease inhibitor mix (Roche Diagnostics, Inc., India- differences between the two groups (68.45⫾4.34 vs.
napolis, IN, USA). CCK release was measured by RIA (37). 71.32⫾6.39, n⫽4). To directly examine the role of
CD36 in FA-induced release of secretin and CCK inde-
Measurement of cAMP, PKA activation, and pendent of fat absorption, intestinal segments were
intracellular Ca2ⴙ
isolated from WT and CD36⫺/⫺ mice and tested for
FA-induced peptide release. Secretin and CCK release
Intracellular cAMP was measured using the DetectX Direct
High Sensitivity Cyclic AMP Chemiluminescent Immunoassay
were similar in WT and CD36⫺/⫺ segments under basal
Kit (Arbor Assays, Ann Arbor, MI, USA). In brief, serum- conditions (data not shown). Addition of 100 and 300
starved STC-1 cells (5⫻105/well) were stimulated with FA M LA enhanced CCK release from WT segments by
(100 M) complexed with BSA (5 M) or BSA alone (30 min, 2.3- and 3.6-fold above basal. No enhancement was
observed in CD36⫺/⫺ segments with 100 M LA, and a lial CD36 expression was down-regulated in intestines
1.6-fold enhancement occurred at 300 M. Secretin from fed (Supplemental Fig. S2C, D) compared with
release from WT segments increased 3.3- and 4.3-fold unfed mice (Supplemental Fig. S2A, B). CD36 was
above basal in response to 100 and 300 M LA. In expressed on nonepithelial cells throughout the small
contrast, the same LA concentrations increased re- intestine, and nonepithelial expression also declined
lease only 1.4- and 1.8-fold in CD36⫺/⫺ segments. with feeding (Supplemental Fig. S2).
These data suggested a major role of CD36 in We next examined expression of CD36 on EECs.
fat-induced EEC secretion of CCK and secretin that Chromogranin A is an essential component of secretory
was independent of the effects of CD36 on luminal vesicles typical of EECs and a marker for most cell types
fat absorption. of the enteroendocrine lineage (41). We costained for
chromogranin A and CD36 in intestinal sections and
CD36 expression in the small intestine and found that a subset of chromogranin A-positive cells
colocalization with chromogranin A express CD36 in the proximal (Fig. 2A) and distal small
CD36 is expressed apically on enterocytes of the small intestine (data not shown), as determined by merge of
intestine (39) after a proximal to distal decreasing fluorescent signals. To quantify the distribution of
gradient (24, 25), and its levels are down-regulated by CD36 across chromogranin A-positive cells, doubly
small amounts of dietary fat (40). Information about positive cells (for chromogranin A and CD36) were
CD36 expression on EECs and whether it is regulated counted and expressed as a percentage of cells positive
by dietary fat is unavailable so this was examined. for chromogranin A only. Sections from 5 mice were
Consistent with previous observations, CD36 was abun- used for quantification. This process demonstrated that
dant on epithelial cells of the duodenum and jejunum ⬃35 and 16% of chromogranin A-positive EECs in the
and less abundant in the ileum (Supplemental Fig. proximal and distal small intestine, respectively, stained
S1A⫺C). No CD36 signal was detected in intestines for both CD36 and chromogranin A (Table 2). Images
from CD36⫺/⫺ mice (Supplemental Fig. S1D). Epithe- 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 pressed CD36 (Table 2 and Fig. 2E). Secretin-positive
small intestine. cells in these sections were also positive for chromo-
granin A (data not shown). EECs project their apical
CD36 is expressed on subpopulations of EECs membrane into the intestinal lumen, and all secretin-
positive EECs may express CD36 on their apical mem-
Our in vivo data showed that CD36 deletion reduced brane (Fig. 2B, C; arrows).
release of secretin and CCK. We examined whether Colocalization of CD36 and CCK was studied using trans-
CD36 expression can be detected on the subpopula- genic mice expressing the GFP driven by the CCK promoter.
tions of EECs involved in release of these peptides. The A small subset of CCK-secreting cells (⬃5%) exhibited
CD36 signal was detected in the cytoplasm of a subset of intracellular costaining for CD36 (Fig. 3A, B and Table 2).
secretin-producing cells in the proximal (Fig. 2B, C) Based on immunofluorescence staining, CCK-positive cells
and distal (data not shown) small intestines. Approxi- may have apical CD36 expression (Fig. 3C, D; arrows).
mately 21 and 9% of secretin-positive cells in the
proximal and distal small intestine, respectively, ex- CD36 enhances secretin and CCK release by STC-1 cells
calcium was also used. The C terminus of CD36 is DHA (Fig. 5A) and LA (Fig. 5B) induced almost 3-fold
important for signal transduction (44, 45). In the enhancement of CCK release, whereas PA (Fig. 5D)
CD36K/A mutant lysines K469 and K472 in the C increased it by approximately 2-fold. OA was still inef-
terminus were substituted with alanine. As we reported fective. No enhancement was observed in cells express-
previously, the CD36K/A mutant has lost the ability to ing vector or CD36K/A. The specificity of CD36 in-
regulate calcium influx, calcium-induced phospho- volvement was validated further by SSO producing
lipase activation, and translocation to membranes (20), almost complete inhibition of CCK release (Fig. 5E).
but it has normal FA uptake activity (38). We hypothe-
sized that this mutant might have impaired regulation Enterocyte coculture on secretin and CCK release by
of granule exocytosis as a result of its defect in calcium STC-1 cells
signaling. All cells were treated with 50, 100, or 200 M
FA (bound to 5 M BSA), and the effect on peptide Our experiments using intestinal segments in vitro
secretion was monitored. suggested that CD36 regulation of EEC release of
Secretin release was induced 2-fold above basal by 50 secretin and CCK was independent of changes in fat
M DHA, in CD36-expressing STC-1 cells, and no absorption (Fig. 1). To examine this result further we
enhancement was observed in cells expressing either tested whether CD36 expression on enterocytes influ-
CD36K/A or the empty vector (Fig. 4B). Similar data ences peptide secretion by neighboring EECs. Studies
were observed with LA (Fig. 4C) and linolenic acid were conducted using coculture of STC-1 cells with
(data not shown). OA and PA were ineffective (Fig. 4D, Caco-2 cells, a well-studied model of enterocytes (47–
E). As the FA concentration was increased to 100 M, 49). Differentiated Caco-2 cells with stable expression
secretin release increased to 3.5 times basal with DHA of CD36, which are responsive to FA signaling (data not
(Fig. 4B) and to 3 and 2 times basal with LA (Fig. 4C) shown), were generated and used for these experi-
and OA (Fig. 4D), respectively. However, modest en- ments. Cocultures of Caco-2 and STC-1 cells, with (⫹)
hancement of release (1.5–1.8 times basal) could now or without (⫺) stable expression of CD36 were tested
be observed in control (vector and CD36K/A) cells. At for LA-induced release of secretin and CCK. At 50 M,
200 M FA, the stimulatory effects of DHA, LA, and OA LA enhanced secretin release in STC-1 cells stably
on secretin release were slightly more than those at 100 expressing CD36 (⫹), compared with that in empty
M, and PA had a small effect. To further validate the vector controls (⫺) by⬃2-fold above basal whether the
CD36 specificity of observed effects, the irreversible coculture contained Caco-2 cells expressing CD36 (⫹)
CD36 inhibitor sulfo-N-succinimidyl oleate (SSO; ref. or empty vector (⫺) (Fig. 4G). A similar trend was
46) was used. In cells preincubated with SSO (20 M, observed with 200 M LA. Secretin release increased
15 min) and then treated with 100 M FA, secretin similarly and ⬃4-fold over basal in cocultures contain-
release was blunted (Fig. 4F), whereas no significant ing STC-1 (⫹) cells whether the cocultured Caco-2 cells
effect was observed on CD36-independent release (vec- did or did not express CD36. In contrast with 50 M
tor and CD36K/A-expressing cells). LA, 200 M LA enhanced secretion ⬃2-fold in STC-1
cells not expressing CD36 (Fig. 4G). For CCK release,
CCK the trends were similar to those observed with secretin
(Fig. 5F). LA at 50 and 200 M stimulated CCK release
DHA at 50 M stimulated CCK release by 2-fold from by 2- to 2.2- and 2.7- to 2.9-fold, respectively, in STC-1
cells expressing CD36 with no effect on empty vector or cells stably expressing CD36 (⫹) compared with that in
CD36K/A-expressing cells (Fig. 5A). Similar effects vector controls (⫺), independent of CD36 expression
were observed with LA (Fig. 5B). PA induced a modest in Caco-2 cells. These data suggested limited contribu-
30% increase (Fig. 5D), whereas OA was ineffective tion of CD36 expression on adjacent enterocytes to
(Fig. 5C). At higher concentrations of FA (200 M), FA-stimulated peptide release.
1196 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org 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.
cAMP production is involved in CD36-dependent no significant effect on release of these peptides in the
peptide release CD36K/A mutant. These data implicated cAMP gener-
ation in CD36-mediated FA-induced release of secretin
cAMP regulates a variety of secretory events including and CCK.
EEC release of secretin and CCK (30, 50). Its role in
CD36-mediated effects was examined. LA treatment CD36-mediated secretin release is dependent on PKA
increased intracellular cAMP (⬃50%) compared with activation
that in BSA controls, and this effect was strongly
amplified in CD36-expressing cells in which cAMP A major effector of cAMP is PKA, which, when acti-
increased by 240% (Fig. 6A). DHA increased cAMP vated, phosphorylates an array of protein substrates
(⬃88%) only in these cells, whereas PA had no effect (51). We determined the effect of FA on PKA activity by
(Fig. 6A). Further, the cAMP inhibitor MDL12230A (10 measuring phosphorylation of its substrates (RRXS*/T*
M, 30 min) reduced CD36-dependent release of both motifs). Addition of LA (Fig. 7A) or DHA (data not
secretin (Fig. 6B) and CCK (Fig. 6C). MDL12230A had 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- in Chinese hamster ovary, macrophages, and taste bud
tion of PKA substrates in STC-1 cells expressing WT cells (20, 22). We investigated effect of CD36 expres-
CD36 but not in cells expressing the signaling impaired sion on calcium transients in response to LA in the
mutant CD36K/A (Fig. 7A). Under the same condi- absence or presence of extracellular calcium. A rapid
tions, PA treatment was ineffective (Supplemental Fig. increase in intracellular calcium was observed in LA-
S3). Activation of the PKA tetramer associates with treated STC-1 cells expressing CD36 WT on addition of
translocation of the catalytic subunit to the nucleus medium calcium, whereas no effect was observed in
(51). LA treatment induced nuclear translocation of cells expressing the vector or CD36K/A mutant (Fig.
catalytic PKA C-␣ in control cells, and this effect was 7E). Calcium influx activates CaM-KII, which is involved
amplified 3-fold in cells expressing CD36 WT (Fig. 7B). in release of CCK (32). Indeed, treatment with the
LA increased nuclear protein kinase C content by CaM-KII inhibitor KN-62 (2.5 M, 15 min) blunted
⬃2.4-fold in vector cells, by 7.4-fold in CD36WT cells, CCK release in CD36-expressing cells (Fig. 7F), sup-
and by 1.7-fold in CD36K/A cells. The responses of porting involvement of CaM-KII in CD36-mediated
vector and CD36K/A-expressing cells were similar and CCK release.
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- DISCUSSION
pressing cells (Fig. 7C), suggesting that release involved
PKA activation. In contrast, CD36-mediated, FA-in- CD36 contributes to different steps of the fat absorp-
duced CCK release was not affected by H-89 (Fig. 7D), tion process (53); it mediates fat perception in taste
implicating PKA-independent mechanisms downstream of bud cells and the initiation of the cephalic phase of
cAMP. digestion (22). It also influences intestinal fat process-
ing and chylomicron formation (25, 26, 28). This study
CD36-mediated CCK release involves CaM-KII documented a novel and potentially important meta-
bolic function of CD36: its mediation of gut peptide
An increase in intracellular calcium after cAMP gener- release in response to dietary FA. We showed that CD36
ation has been implicated in release of CCK (50, 52). is expressed on EECs positive for secretin and CCK and
CD36 was recently reported to influence calcium flux the release of these peptides in response to intragastric
1200 Vol. 27 March 2013 The FASEB Journal 䡠 www.fasebj.org SUNDARESAN ET AL.
homeostasis (12, 13). Regulation of FA-induced CCK mice through the activation of the melanocortin system. Neuro-
and secretin release contributes to the role of CD36 in psychopharmacology 36, 459 –471
13. Dockray, G. J. (2012) Cholecystokinin. Curr. Opin. Endocrinol.
intestinal handling of dietary fat. In humans, common Diabetes Obes. 19, 8 –12
SNPs in the CD36 gene influence plasma lipids (59), 14. Hajri, T., and Abumrad, N. A. (2002) Fatty acid transport across
and CD36 deficiency is characterized by altered chylo- membranes: relevance to nutrition and metabolic pathology.
Annu. Rev. Nutr. 22, 383–415
micron production (60). In addition, SNPs that reduce 15. Oh, D. Y., and Lagakos, W. S. (2011) The role of G-protein-
the CD36 level associate with diminished oral FA sen- coupled receptors in mediating the effect of fatty acids on
sitivity (23). Whether intestinal FA sensing is also inflammation and insulin sensitivity. Curr. Opin. Clin. Nutr.
altered in these subjects remains to be determined. FA Metab. Care 14, 322–327
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The authors acknowledge valuable assistance by Terri 18. Oh, D. Y., Talukdar, S., Bae, E. J., Imamura, T., Morinaga, H.,
Pietka (Adipocyte Biology and Molecular Nutrition Core, Fan, W., Li, P., Lu, W. J., Watkins, S. M., and Olefsky, J. M.
Nutrition and Obesity Research Center, Washington Univer- (2010) GPR120 is an omega-3 fatty acid receptor mediating
sity School of Medicine; P30-DK056341) and are grateful to potent anti-inflammatory and insulin-sensitizing effects. Cell
Dr. Ondrej Kuda for help with the calcium flux studies and to 142, 687–698
Dr. Doug Hanahan for the permission to obtain STC-1 cells 19. El-Yassimi, A., Hichami, A., Besnard, P., and Khan, N. A. (2008)
Linoleic acid induces calcium signaling, Src kinase phosphory-
from American Type Culture Collection (Manassas, VA, lation, and neurotransmitter release in mouse CD36-positive
USA). This work was supported by funding from U.S. Na- gustatory cells. J. Biol. Chem. 283, 12949 –12959
tional Institutes of Health grants DK033301, DK60022 (to 20. Kuda, O., Jenkins, C. M., Skinner, J. R., Moon, S. H., Su, X.,
N.A.A.), DK33165, DK55753 (to W.F.S.), and DK091946 (to Gross, R. W., and Abumrad, N. A. (2011) CD36 protein is
R.A.L.). R.S., T.E.R., R.C., and F.N. assisted in data acquisition involved in store-operated calcium flux, phospholipase A2 acti-
and reviewed the manuscript; W.F.S. and R.A.L. critically vation, and production of prostaglandin E2. J. Biol. Chem. 286,
reviewed the manuscript; S.S. obtained and analyzed data; 17785–17795
and S.S. and N.A.A. were involved in development of the 21. Khan, N. A., and Besnard, P. (2009) Oro-sensory perception of
study concept and design, data analysis and interpretation, dietary lipids: new insights into the fat taste transduction.
Biochim. Biophys. Acta 1791, 149 –155
and manuscript preparation. 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
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