From the Departments of a Metabolic Diseases, h Systems Research, d Genomic Histology, k Cellular Genomics, and n Cell Physiology, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, the Departments of e Vascular Biology, l Systems Research, i Quantitative Expression, and g Gene Cloning, New Frontiers Science Park, GlaxoSmithKline, Southern Way, Harlow, Essex CM19 5AD, United Kingdom, and the Departments of f Gene Cloning and m Expression Genomics, GlaxoSmithKline, King of Prussia, Pennsylvania 19406
Received for publication, November 11, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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GPR40 is a member of a subfamily of
homologous G protein-coupled receptors that include GPR41 and GPR43 and
that have no current function or ligand ascribed. Ligand fishing
experiments in HEK293 cells expressing human GPR40 revealed that a
range of saturated and unsaturated carboxylic acids with carbon chain
lengths greater than six were able to induce an elevation of
[Ca2+]i, measured using a fluorometric
imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent
fatty acid tested, with a pEC50 of 5.7. G protein coupling
of GPR40 was examined in Chinese hamster ovary cells expressing the
G G protein-coupled receptors (GPCRs)1 not only respond
to a large variety of molecules from
inorganic ions to peptides but are also critical for a diversity of
physiological functions. In the last decade, an increasing number of
unliganded receptors, so-called orphan receptors, with unknown function
have been identified, warranting much research into their biological
role. GPR40 was cloned along with GPR41-43
downstream of CD22 on human chromosomal locus 19q13.1 (1). GPR40-43
belong to a subset of orphan receptors, which are more related to each
other than to other liganded receptors, with GPR40 being 30% identical
to GPR41 and GPR43. The group of GPCRs of which GPR40-43
are members is thought to have evolved relatively recently and contains
several gene duplications, of which GPR41 and
GPR42 are examples (2).
Fatty acids play an important physiological role in many tissues. Many
effects attributable to fatty acids, such as impairment of
insulin-mediated glucose uptake and glycogen synthesis in muscle (3)
and potentiation of glucose-stimulated insulin secretion in pancreatic
islets (4), have been attributed to their intracellular metabolism to
long chain acyl-CoA esters. However, it is also conceivable that fatty
acids may act at a cell-surface receptor because receptors for fatty
acid derivatives such as prostaglandins and leukotrienes have been
identified (5, 6). Moreover, there is significant homology between the
GPR40-43 receptor family and the leukotriene receptor family.
However, to date, no extracellular receptor for the fatty acids most
prevalent in plasma (palmitate, oleate, stearate, linoleate, and
linolenate) has been reported.
Using a ligand fishing strategy, previously successful in identifying
ligands for other orphan GPCRs (7), we demonstrate that medium and long
chain saturated and unsaturated fatty acids can activate GPR40 in a
dose-dependent manner. However, short chain fatty acids,
shown to activate other family member receptors (GPR41 and GPR43), are
inactive in cells expressing GPR40 (2). Pharmacological analysis of
GPR40 and its tissue distribution suggests that the receptor may play a
role in pancreatic Cloning of GPR40--
The human GPR40
(hGPR40) receptor (GenBankTM/EBI accession
number AF024687) was cloned by PCR from human genomic DNA using nested
oligonucleotide sense primers (5'-GATCTGAGGACAGGGAGCCAGGTTGCA-3' and 5'-GGAGCCGTGCAGGCC AGGACG-3') and antisense primers
(5'-CCCTGCAGTTCCTCCGAAGCAGC-3' and 5'-CATGCTCCTTCCCCCGAGCAG-3')
designed based on the genomic sequence of the hGPR40 gene.
Mouse Gpr40 was cloned by hybridization of the
32P-radiolabeled human coding region to a mouse genomic
phage library (Stratagene, La Jolla, CA) under low stringency
conditions as previously described (8). Positive clones were
plaque-purified, digested with BamHI, and size-fractionated
by agarose gel electrophoresis. Southern blot analysis with the
hGPR40 probe was used to identify a BamHI fragment that was subcloned into pBluescript (Stratagene). The subclones were sequenced (PerkinElmer Life Sciences); and one clone,
designated mouse Gpr40g, contained an open reading frame of
300 amino acids with 83% identity to hGPR40. Oligonucleotides were
designed to clone mouse Gpr40 from brain cDNA using
Pfu Turbo (Stratagene). A sequence confirming the cDNA
clone (GenBankTM/EBI accession number AF539809) was
subcloned into the mammalian expression vector pCDN (9).
Rat Gpr40 was cloned using oligonucleotides designed in the
regions of high homology between the human and mouse genes. A partial
fragment of 435 bp was cloned from rat pancreatic and brain cDNAs
and sequenced (PerkinElmer Life Sciences). Following confirmation that
the clone was the rat ortholog to human and mouse genes, 5'- and
3'-RACE oligonucleotides were designed, and a full-length rat
Gpr40 cDNA (GenBankTM/EBI accession number
AF539810) was cloned using Pfu Turbo. A sequence confirming
the cDNA clone was subcloned into pCDN (9).
Cell Culture--
CHO cells were maintained in Dulbecco's
modified Eagle's medium/nutrient mixture F-12 (Invitrogen)
containing 5% fetal bovine serum (v/v) and 2 mM glutamine.
MIN6 cells (a gift from Professor J. Miyazaki (University of Tokyo,
Japan) through Professor Kevin Docherty (University of Aberdeen,
Aberdeen, UK)) (10) were grown in Dulbecco's modified Eagle's
medium (Invitrogen) containing 15% (v/v) fetal calf serum, 25 mM glucose, 2 mM glutamine, and 50 µM Stable Expression of GPR40 in Mammalian
Cells--
hGPR40 in pCDN was stably expressed in HEK293
cells using LipofectAMINE Plus (Invitrogen) according to the
manufacturer's instructions. Stable clonal cell lines were generated
by serial dilution into growth medium containing 400 µg/ml G418
(Invitrogen). Stable CHO cells expressing hGPR40, a
chimeric Gal4-Elk1 transcription factor, and a five-Gal4 promoter
upstream of a luciferase reporter gene
(hGPR40-Gal4-Elk1/5xGal4-luc+ cells) were generated as
follows. hGPR40 was expressed in stable Gal4-Elk1/5xGal4-luc+-expressing CHO cells (host cells)
essentially as described (11) using a Gal4-Elk1/5xGal4-luc+
puromycin vector (a gift from Erik Whitehorn, Affymax Corp., San
Francisco, CA). Clones were selected in medium containing puromycin.
After fluorescence-activated cell sorting and plating individual cells
into 96-well plates, clones were chosen by response to thrombin
(Sigma). 12 µg of hGPR40 vector was subsequently
transfected into the Gal4-Elk1/5xGal4-luc+ host cells
essentially as described (11), except that clones were selected in 500 µg/ml G418 and chosen by response to 5,8,11-eicosatriynoic acid (ETA) (Sigma).
Calcium Mobilization Assays--
HEK293 cells transiently or
stably expressing GPR40 were seeded (50,000 cells/well) into
poly-D-lysine-coated 96-well black-view, clear-bottom microtiter plates (BD Biosciences, Oxford, UK) 24 h
prior to assay. Cells were loaded for 1 h with 1 µM
Fluo-4/AM fluorescent indicator dye (Molecular Probes, Inc.,
Leiden, Netherlands) in assay buffer (Hanks' balanced salt solution,
10 mM HEPES, 200 µM Ca2+, 0.1%
bovine serum albumin, and 2.5 mM probenecid), washed three times with assay buffer, and then returned to the incubator for 10 min
before assay on a fluorometric imaging plate reader (FLIPR) (Molecular
Devices, Wokingham, UK). Maximum change in fluorescence over the base
line was used to determine agonist response. Cells were screened
against a large library of >1500 known and putative GPCR agonists,
including all known mammalian neuropeptides (tachykinins, neuromedins,
etc.), bioactive lipids (leukotrienes, prostaglandins, etc.), steroids
(aldosterone, testosterone, etc.), amines (catacholamines, etc.),
cannabinoids (anandamide, etc.), nucleotides (ATP, ADP, UTP, etc.), and
sugar nucleotides (UDP-glucose, UDP-galactose, etc.). Peptides were
tested at a final concentration of >100 nM and other
ligands at >1 µM. Free fatty acids were purchased from Sigma. Concentration-response curve data were fitted to a
four-parameter logistic equation using GraFit (Erithacus Software
Ltd.).
96-Well Adherent Reporter Cell Assay--
The reporter assay for
the stable hGPR40-Gal4-Elk1/5xGal4-luc+-expressing
CHO cells was carried out as follows. Forty-eight hours prior to assay,
hGPR40-Gal4-Elk1/5xGal4-luc+ cells were plated at a
concentration of 10,000 cells/well in a 96-well black-view plate
(Costar Corp.) in complete medium. Eighteen hours prior to the assay,
the medium was replaced with 90 µl/well serum-free complete medium
with or without 1 µg/ml pertussis toxin (PTX) (Calbiochem). ETA was
made at 20 mM in 100% ethanol with subsequent dilutions
accompanied by sonication. Oleoyllysophosphatidic acid (Sigma) was made
in water. At the time of the assay, the medium was replaced with 90 µl of serum-free medium; 10 µl of ligand was added; and plates were
incubated for 5 h at 37 °C. Subsequently, the medium was
removed and replaced with 50 µl of a 1:1 mixture of
LucPlusTM (PerkinElmer Life Sciences) and Dulbecco's
phosphate buffered saline containing 1 mM
CaCl2 and 1 mM MgCl2. Plates were
incubated in the dark at room temperature for 10 min before luciferase
activity was determined using a TopCountTM microplate
scintillation counter (PerkinElmer Life Sciences) using a count time of
3 s/well.
Preparation of Islets of Langerhans--
Rat islets were
prepared by a collagenase method (12) from the pancreases of Wistar
rats. Batches of human islets were isolated from heart-beating adult
cadaver organ donors with informed consent and supplied by the Human
Islet Facility at the University of Leicester (Leicester, UK).
Localization Studies--
TaqMan quantitative PCR was performed
on 1 ng of cDNA prepared from human tissues (Promega, Madison, WI)
essentially as described previously (6), using forward and
reverse hGPR40-specific primers and probes
(BIOSOURCE, International, Camarillo, CA). Primers and probes were designed using Primer Express software (PE Applied Biosystems, Foster City, CA) and are shown in Table I. Copy number was
calculated from a standard curve, generated using sheared genomic DNA,
assuming that 1 µg/µl sheared genomic DNA is equivalent to 6 × 105 "single-strand copies." The data are presented
as mRNA copies detected per ng of poly(A)+ RNA from
four individuals (means ± S.E., n = 4). For the
immune cell samples, TaqMan analysis was performed on 50 ng of total RNA extracted from samples from up to three individuals as described (2). Relative abundance in these samples is expressed as
2
Total RNA was isolated from human, rat, or mouse islets or MIN6 cells
using TRIzol (Invitrogen), and cDNA was generated as previously
described (13). Expression of GPR40 in islets and MIN6 cells was
carried out using species-specific TaqMan primers/probes (BIOSOURCE, International), and copy number was
calculated using a standard curve created using rat or mouse
Gpr40 cDNA. Pancreatic RNA was extracted from
20-week-old ob/ob and lean mice by acid guanidine/thiocyanate/phenol/chloroform extraction, with expression of
GPR40 in pancreas performed using TaqMan primers and probes (PE
Applied Biosystems) designed across mouse and rat Gpr40
genes (Table I).
In Situ Hybridization Experiments--
A 587-base probe was
generated by reverse transcription-PCR from the full-length clone of
rat Gpr40 and subcloned into the transcription vector pGEM-T
(Promega). After cloning, the vector was linearized with the
restriction enzyme SpeI, followed by the generation of
radiolabeled antisense transcripts using T7 RNA polymerase and
[33P]ribonucleotideUTP (800 Ci/mol; Amersham
Biosciences). Sense control probes were generated by linearization with
NcoI, followed by transcription with SP6 polymerase.
Whole rat pancreas was obtained by rapid necropsy, fixed in neutral
buffered 10% Formalin for 24 h, and embedded in paraffin. The
tissues were sectioned at 6 µm, mounted on plus slides,
deparaffinized, rehydrated, and then pretreated with 0.2 M
HCl for 10 min, followed by digestion with 10 µg/ml proteinase K for
20 min. After dehydrating and drying, the sections were prehybridized
for 2 h in 50% prehybridization mixture (600 mM NaCl,
40 mM Tris (pH 8.0), 10 mM EDTA, 2×
Denhardt's solution, 0.4% SDS, 20 mM dithiothreitol, and
0.5 µg/ml tRNA) and 50% formamide at 55 °C. The probes (2 × 106 cpm/section) were mixed with hybridization solution
(2× hybridization mixture and 20% dextran sulfate in formamide, 1:1
(v/v)) and hybridized overnight at 55 °C. The following morning, the
sections were then washed at 55 °C with 5× SSC for 1 h and
with 0.1× SSC for 30 min, digested with RNase A (20 µg/ml) at
37 °C for 30 min, washed again with 0.1× SSC at 55 °C for 30 min, and dehydrated. After drying, the slides were dipped in Kodak
NTB-2 emulsion, exposed for 4 weeks, developed with Kodak
D-19 developer, counterstained with hematoxylin, and
examined by both dark-field and light-field microscopy. Immunohistochemical staining for insulin, glucagon, and somatostatin was performed using a Ventana Nexus automated stainer with all Ventana
reagents (Ventana Medical Systems, Inc., Tucson, AZ). An enhanced
3,3'-diaminobenzidine kit was used for detection, and sections
were counterstained with hematoxylin.
Statistical Analysis--
For each concentration of ligand used
in reporter assays, one-way analysis of variance was performed.
Comparison of pEC50 values was performed using Student's
t test. p < 0.05 was considered statistically significant.
Activation of hGPR40 Expressed in HEK293 Cells--
We transiently
transfected HEK293 cells with hGPR40 and screened these cells for their
ability to increase intracellular calcium using a FLIPK against a large
library of >1500 known and putative natural GPCR agonists. Elaidic
acid (C18H34O2) was the only ligand found in the library to specifically activate the receptor compared with cells transiently expressing other GPCRs (Fig.
1A). Further testing of other
fatty acids in HEK293 cells stably expressing GPR40 demonstrated that a
range of medium to long chain saturated and unsaturated fatty acids,
e.g. elaidic acid, palmitic acid, and ETA, were able to
increase intracellular calcium in a concentration-dependent manner, whereas short chain fatty acids with carbon chain lengths greater than six, e.g. succinic acid and formic acid,
produced no response (Fig. 1B).
pEC50 values for both saturated and unsaturated fatty acids for the elevation of intracellular calcium in HEK293 cells
are shown in Table I. The most potent
saturated fatty acids were those with carbon chain lengths of 15 and
16, whereas of all the fatty acids tested, ETA was the most potent,
with a pEC50 of 5.71.
Activation of the
Gal4-Elk1/5xGal4-luc+ Reporter in Cells
Expressing hGPR40--
We stably transfected hGPR40 into a stable CHO
cell line containing a Gal4-Elk1/5xGal4-luc+ reporter.
Following activation of MAPK, the chimeric Gal4-Elk1 transcription
factor is phosphorylated and activated and binds to the Gal4 upstream
activating sequence, resulting in induction of luciferase expression
(14). MAPK activation can occur downstream of signaling pathways
initiated by receptors coupled to either G
To investigate the G protein coupling of GPR40 following ligand binding
and activation, hGPR40-Gal4-Elk1/5xGal4-luc+ cells
were treated with the fatty acid ETA, the most potent ligand for hGPR40
from the initial ligand pairing screen performed in HEK293 cells (Table
I). As a positive control, cells were also treated with
lysophosphatidic acid (LPA), which binds to an endogenous G Expression Profile of Human, Mouse, and Rat mRNAs--
To
evaluate the physiological role of GPR40, we performed real-time
quantitative reverse transcription-PCR using TaqMan in a variety of
human tissues from one to four individuals. Specific expression was
observed in pancreas and brain (Fig. 3).
Further analysis of expression of hGPR40 in brain
regions revealed ubiquitous expression in the areas examined, with the
highest expression in the substantia nigra and medulla oblongata.
Analysis of GPR40 expression in immune cells from one to
three individuals showed that, in contrast to hGPR43, no
expression of GPR40 was detected in peripheral blood
mononuclear cells, B-lymphocytes, or neutrophils (2). Expression of
GPR40 was detected in monocytes, although at relatively low
levels compared with that of GPR43. Because the analysis of
GPR40 and GPR43 expression was performed in the same immune cell samples, the data show that there is clear
differential expression between these two receptors in immune cells.
As the pancreas is composed of exocrine cells and endocrine islet
cells, with the islets making up only 2% of the pancreas, the level of
hGPR40 expression in the pancreas was compared with that in
human islets. In four independent islet samples, there was a
2-100-fold higher expression of GPR40 than in total
pancreas for an equivalent RNA input, with a mean -fold increase in
expression of 36.8 ± 25.4 compared with that in total pancreas.
These data suggest that pancreatic expression of GPR40 may
be localized to human pancreatic islets. Islets contain four cell types
( We have identified medium and long chain saturated and unsaturated
fatty acids as ligands for the orphan G protein-coupled receptor GPR40
using increases in [Ca2+]i, measured using FLIPR
in HEK293 cells expressing GPR40. Fatty acids have also been previously
identified as natural ligands for the nuclear receptor peroxisome
proliferator-activated receptor- The potency of the saturated fatty acids was chain
length- dependent, with pentadecanoic acid
(C15) and palmitic acid (C16) being the most
potent, with pEC50 values of 5.18 ± 0.11 and
5.30 ± 0.12, respectively. Across the unsaturated fatty acids,
potency did not appear to correlate with carbon chain length or degree of saturation. Of all the fatty acids tested, both saturated and unsaturated, ETA was the most potent, with a pEC50 of
5.71 ± 0.11. The potencies for GPR40 of the fatty acids most
predominant in plasma (oleate, palmitate, stearate, linoleate, and
linolenate) are at reported physiologically relevant concentrations
(20). However, whether GPR40 agonism by some of the fatty acids present at much smaller proportions of the total non-esterified fatty acids is
important in the body is not clear.
ETA is commonly used as an inhibitor of 5- and 12-lipoxygenases in the
range of 2-100 µM. ETA has also been reported to
increase intracellular calcium in Madin-Darby canine kidney cells, with an EC50 of 20 µM, although treatment with the
phospholipase C inhibitor U73122 suggested that this may not be due to
production of inositol 1,4,5-trisphosphate (21). Expression of GPR40 in Madin-Darby canine kidney cells is unknown; and hence, whether the
reported mobilization of [Ca2+]i or inhibition of
lipoxygenase activity is mediated through GPR40 activation remains to
be elucidated.
Given the number of fatty acids that are agonists for GPR40, it is
conceivable that the physiologically relevant fatty acid for GPR40 may
vary in a tissue-dependent fashion. Notably,
several of the fatty acids shown to activate GPR40 have also been
reported to activate a chimera of peroxisome proliferator-activated
receptor- Experiments using CHO cells expressing the
Gal4-Elk1/5xGal4-luc+ reporter, responsive to
G TaqMan RT-PCR showed that GPR40 was specifically expressed
in human brain and pancreas, with no expression detected in resting immune cells, apart from low levels in human monocytes. This pattern of
expression clearly differentiates GPR40 from the other
family members GPR41 and GPR43 (2), suggesting
that its function has clearly diverged from that of its related receptors.
The pancreas is composed of exocrine and endocrine tissue. The
endocrine cells or islets make up 1-2% of the pancreas. Comparison of
mRNA expression abundance by TaqMan analysis illustrated that GPR40 was enriched in islets by 2-100-fold compared with
the pancreas and that the receptor was present in isolated islets from
mouse and rat pancreases. Islets contain four cell types:
glucagon-producing Fatty acids are recognized to play an important role in maintenance of
basal insulin secretion and potentiation of glucose-stimulated insulin
secretion in the fasting state in both rodent and human islets
(22-25). To stimulate insulin secretion, glucose enters the We report that the GPR40 agonist ETA activated a
G Prolonged exposure to elevated free fatty acids, which may occur in
obesity or in states of insulin resistance, has, however, been found to
be detrimental to Further clarification of the role of GPR40 in GPR40 is also expressed ubiquitously in human brain, with
the highest expression in the substantia nigra and medulla oblongata. Notably, docosahexaenoic acid (DHA), known since the 1960s (38) to be
present at a high level in mammalian brain, had a pEC50 of
5.37 for GPR40. Not only is DHA known to be essential for neural development and function (39); but more recently, enrichment of DHA in
cell membranes has been found to have an anti-apoptotic effect on
neural apoptosis (40). Although apoptosis is a critical part of neural
development, it also plays a fundamental role in the neurological
disorders of aging, viz. Alzheimer's disease, Parkinson's
disease, and stroke (41). The mechanism behind the anti-apoptotic
effect of DHA is currently unclear, but has been proposed to involve an
increase in phosphatidylserine synthesis and gene expression. Although
DHA tends to be retained by membrane phospholipids, astroglia cells,
known to support neuronal survival, do release DHA, suggesting that
this may reach a local concentration sufficient to act as an
extracellular signaling molecule (29, 42). Further information as to
possible roles of GPR40 in brain function may be obtained by studying
the changes in expression of the receptor in neural development and in
diseased states.
In summary, we have identified fatty acids as ligands for the orphan
receptor GPR40. The distribution of the receptor suggests that, in
addition to their effects mediated through their intracellular metabolism, fatty acids may act as extracellular signaling molecules, at a membrane receptor, to regulate pancreatic islet and neurological function.
q/i-responsive Gal4-Elk1 reporter system. Expression of
human GPR40 led to a constitutive induction of luciferase activity,
which was further increased by exposure of the cells to eicosatriynoic
acid. Neither the constitutive nor ligand-mediated luciferase induction
was inhibited by pertussis toxin treatment, suggesting that GPR40 was
coupled to G
q/11. Expression analysis by quantitative
reverse transcription-PCR showed that GPR40 was
specifically expressed in brain and pancreas, with expression in rodent
pancreas being localized to insulin-producing
-cells. These data
suggest that some of the physiological effects of fatty acids in
pancreatic islets and brain may be mediated through a cell-surface receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell and neurological function. We have
therefore identified a potential novel mechanism by which fatty acids
may elicit cellular responses in certain tissues.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. HEK293 cells were grown in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and 2 mM glutamine. All cell lines were cultured at
37 °C with 5% CO2.
Ct to reflect the exponential nature of PCR.
The
Ct = 40 cycles
the threshold cycle (Ct).
The Ct value is the cycle number at which the reporter
fluorescence, generated by cleavage of the probe, passes a fixed
threshold above the base line. The Ct occurs in the exponential
phase of PCR when none of the reaction components are limiting. We used
an arbitrary cutoff of 40 cycles as a reference. In other words, each
cycle represents an ~2-fold difference in mRNA abundance.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Long (but not short) chain fatty acids cause
concentration-dependent increases in intracellular calcium in
HEK293 cells expressing GPR40. A, HEK293 cells were
transiently transfected with GPR40, and changes in intracellular
calcium levels were measured following challenge with test ligand.
Elaidic acid (35 µM), added at the 10-s time point,
induced a robust transient elevation of intracellular calcium levels in
GPR40-transfected cells (solid line), but not in cells
transfected with a different receptor (dashed line). Data
are expressed as change in fluorescence intensity units
(FIU) over background levels and are from a single
representative experiment. B, HEK293 cells stably expressing
GPR40 were challenged with fatty acids, and changes in
[Ca2+]i were measured. Cells responded in a
concentration-dependent manner to the long chain fatty
acids elaidic acid ( ), palmitic acid (
), and ETA (
). Formic
acid (
), a short chain fatty acid, exhibited no response. Data shown
are the means ± S.E. from a single representative experiment,
with each point determined in triplicate.
pEC50 values of fatty acids tested in HEK293 cells stably expressing
GPR40
i/o or
G
q/11. The signaling pathways involved can, however, be
inferred through the use of PTX, which ADP-ribosylates
G
i/o, preventing its interaction with receptors while
having no effect on G
q/11 (15). The
hGPR40-Gal4-Elk1/5xGal4-luc+ cells exhibited a
significantly higher basal luciferase activity compared with host cells
lacking hGPR40 (p < 0.01) (Fig.
2A). Pretreatment with PTX
decreased basal luciferase activity in both host cells and cells
expressing GPR40 to a similar extent (luciferase activity in
PTX-treated cells as percent of untreated cells (means ± S.E.,
n = three experiments): host cells, 52.9 ± 9; and
GPR40-expressing cells, 44.2 ± 1.2). These data suggest that some
of the basal luciferase activity present in both host cells and
hGPR40-Gal4-Elk1/5xGal4-luc+ cells was due to the
constitutive activity of endogenous G
i/o-coupled receptors. Luciferase activity remained significantly higher in hGPR40-Gal4-Elk1/5xGal4-luc+ cells compared with
host cells even after PTX treatment. This residual activity may be due
to the constitutive activity of GPR40 resulting from coupling to
G
q/11. Similar results in melanophores, showing
the constitutive activity of GPR40 resulting from coupling to
G
q/11, support this finding (data not shown).
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Fig. 2.
CHO cells stably transfected with GPR40 and
the Gal4-Elk1/5xGal4-luc+ reporter exhibit constitutive
activation of luciferase reporter activity, which is
dose-dependently increased by ETA and is not attenuated by
PTX. A, increase in luciferase activity in
hGPR40-Gal4-Elk1/5xGal4-luc+-expressing CHO cells compared
with host cells following overnight treatment with (black
bars) or without (white bars) PTX. Notably, the
attenuation of luciferase activity by PTX treatment as a proportion of
maximum activity was similar in host cells and
hGPR40-Gal4-Elk1/5xGal4-luc+ cells. B, ETA
induction of luciferase activity in
hGPR40-Gal4-Elk1/5xGal4-luc+-expressing CHO cells and host
cells with or without PTX treatment. C, induction of
luciferase activity by LPA in host cells with or without PTX treatment.
, host cells without PTX;
, host cells with PTX;
,
hGPR40-Gal4-Elk1/5xGal4-luc+ cells without PTX;
,
hGPR40-Gal4-Elk1/5xGal4-luc+ cells with PTX.
Preincubation of host cells with PTX abolished the response to LPA,
whereas the response to ETA in hGPR40-Gal4-Elk1/5xGal4-luc+
cells was enhanced. Data in A are shown as means ± S.E. of luciferase counts (c.p.s.) from one experiment
representative of three, with each point determined in quadruplicate.
Due to the changes in basal activity following PTX treatment, results
for B and C are expressed as -fold over the
control, where the control represents the counts resulting from
luciferase activity in the cell line and under the conditions indicated
in the absence of ligand. Data in B and C are
shown as -fold over the control from three independent
experiments (means ± S.E., where n = 3 within
each experiment).
i-coupled receptor (16). All treatments were performed
following an overnight treatment of PTX or vehicle. ETA (3 µM) produced a maximum 3.3-fold response over the control
in hGPR40-Gal4-Elk1/5xGal4-luc+ cells (Fig.
2B). Responses to 3 µM ETA in four other
hGPR40-Gal4-Elk1/5xGal4-luc+ clones characterized
ranged from 3.3- to 7.4-fold (data not shown). No significant induction
of luciferase activity was observed in host cells at any concentration
of ETA tested (Fig. 2B). LPA in the host cells had a
pEC50 of 5.51 ± 0.01 and produced a 13.5-fold response over basal levels at 2.5 µM (Fig.
2C). The response to LPA was essentially abolished by PTX
treatment. However, PTX did not inhibit the induction of luciferase
activity by ETA in hGPR40-Gal4-Elk1/5xGal4-luc+
cells at any concentration tested, nor did PTX treatment significantly change the pEC50 (without PTX, 5.92 ± 0.05; with PTX,
5.99 ± 0.01). Furthermore, there was a trend for an increase in
ETA-stimulated luciferase activity following PTX treatment, suggesting
that the G
q/11-mediated activation of the luciferase
reporter was dampened by a G
i/0 component. Similar
potentiation of the response to ETA was observed in three of four other
hGPR40-Gal4-Elk1/5xGal4-luc+ clones, whereas in the
fourth clone, PTX had no effect (neither inhibitory nor stimulatory) on
ETA-stimulated luciferase activity (data not shown).
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Fig. 3.
hGPR40 is specifically
expressed in human pancreas and brain, as demonstrated by quantitative
reverse transcription-PCR. The cDNA from the reverse
transcription of 1 ng of poly(A)+ RNA from multiple tissues
from four different non-diseased individuals was analyzed by TaqMan
RT-PCR for expression of GPR40. Data are presented as the
means ± S.E. of four individuals mRNA levels for each tissue,
except for the intestine, which is an equal pool of one intestine and
another individual's large intestine. Analyses of mostly peripheral
tissues and brain region tissues were run as separate experiments.
PBMNC, peripheral blood mononuclear cells.
A, expression of GPR40 in human tissues;
B, expression of GPR40 in human brain regions;
C, expression of GPR40 in freshly isolated human
immune cells and cell lines. Raji is a B-cell line; Jurkat is a T-cell
line; and THP1 is a monocytic cell line.
,
,
, and PP), of which the insulin-secreting
-cells make up >80% of the islet. To further investigate the
localization of Gpr40 in islets, rat and mouse
Gpr40 were cloned. At the DNA level, rat and mouse
Gpr40 are 94% identical to each other and 75 and 76%
identical to hGPR40, respectively. At the protein level, rat and mouse GPR40 are 95% identical to each other and 82 and 83% identical to the human receptor, respectively. To investigate how
expression of Gpr40 changes in a rodent model of obesity and insulin resistance, expression in whole pancreases of
ob/ob mice was examined.
ob/ob mice are obese, hyperglycemic, and
insulin-resistant and exhibit
-cell hyperplasia due to the increased
demand on the cells for insulin (17). Using TaqMan primers specific to rat and mouse Gpr40 (Table
II), significant increases in
expression of Gpr40 mRNA were detected in whole
pancreases of ob/ob mice compared with control
lean mice. Although the increases in Gpr40 mRNA
expression were less than those of insulin (-fold change relative to
lean controls (n = two experiments, 5-10
animals/group): Gpr40, 6.5 ± 1.3; and insulin,
10.2 ± 0.9), these data suggest that the increased expression of
Gpr40 may be due in part to the increased
-cell number
manifest in the pancreas of this animal model. To verify the islet
localization of Gpr40, TaqMan RT-PCR was performed
in rat and mouse islets and in a mouse insulinoma cell line (MIN6)
using species-specific primers. Table III
shows that Gpr40 was expressed in both rat and mouse islets
and in MIN6 cells. In situ hybridization using riboprobes to
rat Gpr40 detected Gpr40 mRNA in sections of
rat pancreas in a staining pattern resembling that found in islets
(Fig. 4). When parallel sections were
immunostained for islet hormones, the pattern of Gpr40
staining was most similar to that of insulin, suggesting that the
receptor is in the islet
-cells and not the peripherally staining
-cells. However, whether expression occurs also in
- and PP-cells
cannot be concluded at this stage.
Sequences of TaqMan primers and probes
Gpr40 is expressed in rodent islets and mouse insulinoma cells
View larger version (150K):
[in a new window]
Fig. 4.
Gpr40 is expressed in
-cells of rat islets, as demonstrated by in
situ hybridization. A: left
panel, dark-field photograph showing hybridization of
33P-labeled Gpr40 riboprobe in the top and
bottom center; right panel, bright-field photograph of the
same field showing that the two regions in which expression was found
are islets. B: upper left panel, dark-field image
from A at a higher magnification; upper right
panel, parallel section from the pancreas immunostained with
insulin, illustrating that Gpr40 was expressed in the same
regions of the islet as insulin; lower left and right
panels, parallel pancreatic sections immunostained with glucagon
and somatostatin, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(18). Although GPCRs for fatty acid
derivatives such as prostaglandins and leukotrienes have been
identified (5, 6), to date, no extracellular receptor for the fatty
acids most prevalent in plasma (palmitate, oleate, stearate, linoleate,
and linolenate) has been reported. The discovery of fatty acids as
ligands for GPR40 is in line with the identification of carboxylic
acids as ligands for GPR41 and GPR43, the other members of this
receptor family (2). However, in contrast to GPR41 and GPR43, GPR40 is
activated by fatty acids with carbon chain lengths of more than six.
Although ligand pairing and G protein coupling experiments were
performed with the free fatty acid in the absence of bovine serum
albumin, no effects were observed in cells not containing GPR40,
demonstrating that the response was not merely a detergent effect.
Furthermore, the concentrations used are well below those at which
fatty acids exert detergent effects on cell membranes (19).
and the glucocorticoid receptor in a reporter gene assay
(18). Although there are similarities in the profile of fatty acids that activate the two receptors, including the fact that fatty acids
with carbon chain lengths of less than six were inactive, there were
also clear differences in relative potencies. For example, linolenic
acid at 0.12 mM was the most efficacious activator of the
fatty acids tested against the peroxisome proliferator-activated receptor-
-glucocorticoid chimera, whereas 0.12 mM lauric
acid was much weaker; in contrast, these two fatty acids both had
pEC50 values of 4.9 for GPR40 and the same maximum
efficacies. Furthermore, the induction of [Ca2+]i
in HEK293 cells or luciferase activity in CHO cells was clearly
dependent on the presence of GPR40, as fatty acids did not elicit a
response in cells not expressing the receptor.
i/o- and G
q/11-coupled
signaling, demonstrated that expression of GPR40 resulted in increased
basal levels of luciferase activity. This reporter is activated
following stimulation of MAPK activity, downstream of signals from
G
i- and G
q-coupled pathways, which
converge at Raf (14). The equivalent decrease in basal luciferase
activity in host cells and hGPR40-Gal4-Elk1/5xGal4-luc+
cells following PTX treatment suggests that there may be an endogenous receptor present in both CHO-derived cell lines, producing a component of constitutive G
i-coupled activity. The residual
elevation in luciferase activity in
hGPR40-Gal4-Elk1/5xGal4-luc+ cells compared with host cells
following PTX treatment suggests that this may be due to the
constitutive activity of GPR40 through a G
q/11-coupled
pathway. Treatment of the hGPR40-Gal4-Elk1/5xGal4-luc+
cells with ETA produced a dose-dependent increase in
luciferase activity, with a pEC50 of 5.92 ± 0.05. PTX
treatment did not alter the pEC50 of ETA in
hGPR40-Gal4-Elk1/5xGal4-luc+ cells (without PTX, 5.92 ± 0.05; and with PTX, 5.99 ± 0.01). However, it did elevate the
apparent efficacy of ETA, an effect that reached significance at
concentrations of ETA between 1 and 2.25 µM. These data
suggest that activation of GPR40 by ETA in hGPR40-Gal4-Elk1/5xGal4-luc+ cells is coupled to a
G
q/11 pathway. Moreover, there may be a small
PTX-sensitive inhibitory component to the ETA-mediated activation of
the Gal4-Elk1/5xGal4-luc+ reporter used, the identification
of which is unclear at present. Experiments using CHO cells transiently
transfected with GPR40 and a luciferase reporter containing
cAMP-response elements demonstrated that the receptor was unable to
increase cAMP levels through a G
s-coupled pathway in
response to ETA (data not shown).
-cells, insulin-producing
-cells,
somatostatin-producing
-cells, and pancreatic
polypeptide-producing-cells. The presence of Gpr40 in mouse
insulinoma MIN6 cells suggested that the receptor may be in
-cells.
Furthermore, the increased expression of Gpr40 in pancreases
from ob/ob mice compared with control animals may have been due to the
-cell hyperplasia manifest in this animal model
of obesity. Whether expression of Gpr40 plays an active role
in the development of hyperplasia or is merely a reflection of this
event is unclear. Further verification that Gpr40 mRNA was expressed in islet
-cells was accomplished using in
situ hybridization. The pattern of Gpr40 expression was
characteristic of
-cell rather than
-cell localization compared
with immunohistochemistry results using antibodies to insulin and
glucagon in parallel pancreatic sections. It remains unclear however,
whether Gpr40 may be in pancreatic
- or PP-cells.
-cell
and is metabolized, resulting in an increase in intracellular ATP and a
decrease in ADP levels. The increase in the ATP/ADP ratio closes
cell-surface KATP channels, promoting membrane
depolarization, opening of voltage-sensitive Ca2+ channels,
and activation of insulin granule exocytosis. Glucose also augments, as
well as initiates, the secretion of insulin through a pathway involving
protein kinases A and C (26, 27). The mechanism by which fatty acids
exert their potentiating effects has not yet been precisely defined.
Some studies have suggested that intracellular metabolism of the fatty
acid to its long chain acyl-CoA ester (LC-CoA) is critical for
nutrient stimulation of insulin secretion (4, 28). Increases in LC-CoA
following glucose administration or exposure of cells to fatty acids
have been proposed to potentiate glucose-stimulated insulin exocytosis through a pathway that may involve phospholipid signaling and activation of protein kinase C. Over carbon chain lengths of
C8-C18, the relative insulinotropic efficacy
of fatty acids is positively correlated with chain length and degree of
saturation (22, 23). It remains unclear, however, whether the
fatty acid acts as a carboxylic acid or needs to be converted
intracellularly into its CoA ester to exert its effect (30). The
identification of fatty acids as ligands for GPR40 highlights the
possibility that fatty acids could also exert some of their effects via
an extracellular mechanism. However, the rank order of potency of fatty
acids for GPR40 in HEK293 cells was not equivalent to the reported
relative efficacy of fatty acids for increasing insulin secretion from perfused pancreas (22) or from islets of fasting rats or from human
islets (23). Furthermore, all fatty acids that increased [Ca2+]i in HEK293 cells expressing GPR40 were
equally efficacious. It is possible, however, that the fact that our
studies were performed in the absence of bovine serum albumin, whereas
other studies were performed in the presence of 1% bovine serum
albumin, may be important.
q-coupled pathway in
hGPR40-Gal4-Elk1/5xGal4-luc+ cells, which would be expected
to lead to an increase in [Ca 2+]i and protein
kinase C and MAPK activation. Ligands (e.g. bombesin or
acetylcholine) (31, 32) known to activate G
q-coupled
receptors in islets potentiate glucose-stimulated insulin secretion.
Thus, if the signaling pathway identified in CHO cells were to be
replicated in islets, GPR40 activation may be anticipated to potentiate
the effects of glucose on insulin secretion.
-cell function both in rodents and humans (33,
34). Because signaling pathways downstream of G
q-coupled
GPCRs may include activation of MAPK and subsequent stimulation of
transcriptional activity, it is also plausible that activation of GPR40
may play a role in the chronic rather than the acute effects of fatty
acids. However, recent reports have demonstrated that the ability of
fatty acids to cause apoptosis in the rat
-cell line RIN1046-38 and
human islets is dependent on the degree of saturation.
Whereas palmitic acid and stearic acid were pro-apoptotic when used at
1 mM for 24 h, linoleic acid, palmitoleic acid, and
oleic acid did not cause apoptosis and actually protected the
-cells
from apoptosis resulting from incubation with saturated fatty acids
(35). Because a large range of both saturated and unsaturated fatty
acids are agonists for GPR40, the primary role of the receptor may
therefore not be mediation of the apoptotic effects of fatty acids.
-cells awaits studies
of
-cell function in transgenic mouse models and analysis of
receptor expression in islets from models of diabetes such as
Zucker diabetic fatty (ZDF) rats, which have high levels of circulating fatty acids due to a defect in the leptin receptor. ZDF rat
islets exhibit intrinsic defects in their ability to secrete insulin in
response to glucose (36), which deteriorates progressively as fatty
acids accumulate in the islets, resulting in apoptosis or
"lipotoxicity" (37).
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ACKNOWLEDGEMENTS |
---|
We acknowledge Steven Novick for statistical analysis, Sharon I. Butler and Angela Smith for assistance with cell culture, and Jean-Philippe Walhin for expert technical assistance with TaqMan assays. We thank Dr. R. Ravid (Netherlands Brain Bank) for arrangement/donation of brain tissue.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF539809 and AF539810.
b To whom correspondence should be addressed. Tel.: 919-483-6127; Fax: 919-483-3777; E-mail: cpb15172@gsk.com.
c Present address: Dept. of Metabolic Research, Boehringer-Ingelheim Pharma KG, D-88397 Biberach, Germany.
j Present address: Upstate Ltd., Gemini Crescent, Dundee Technology Park, Dundee DD2 1SW, United Kingdom.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M211495200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GPCRs, G protein-coupled receptors; hGPR40, human GPR40; RACE, rapid amplification of cDNA ends; CHO, Chinese hamster ovary; ETA, 5,8,11-eicosatriynoic acid; PTX, pertussis toxin; MAPK, mitogen-activated protein kinase; LPA, lysophosphatidic acid; DHA, docosahexaenoic acid; FLIPR, fluorometric imaging plate reader; LC, acylCoA, long-chain acyl-CoA; ZDF, Zucker diabetic fatty.
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REFERENCES |
---|
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---|
1. | Sawzdargo, M., George, S. R., Nguyen, T., Xu, S. J., Kolakowski, L. F., and Odowd, B. F. (1997) Biochem. Biophys. Res. Commun. 239, 543-547[CrossRef][Medline] [Order article via Infotrieve] |
2. | Brown, J. A., Goldsworthy, S. M., Barnes, A. A., Eilert, M. M., Tcheang, L., Daniels, D., Muir, A. I., Wigglesworth, M. J., Kinghorn, I., Fraser, N. J., Pike, N. B., Strum, J. C., Steplewski, K. M., Murdock, P. R., Holder, J. C., Marshall, F. H., Szekeres, P. G., Wilson, S., Ignar, D. M., Foord, M., S., Wise, A., and Dowell, S. J (2003) J. Biol. Chem. 278, 11312-11319 |
3. | Thompson, A. L., Lim-Fraser, M. Y. C., Kraegen, E. W., and Cooney, G. J. (2000) Am. J. Physiol. 279, E577-E584 |
4. |
Prentki, M.,
Vischer, S.,
Glennon, M. C.,
Regazzi, R.,
Deeney, J. T.,
and Corkey, B. E.
(1992)
J. Biol. Chem.
267,
5802-5810 |
5. | Coleman, R. A., Smith, W. L., and Narumiya, S. (1994) Pharmacol. Rev. 46, 205-229[Medline] [Order article via Infotrieve] |
6. |
Sarau, H. M.,
Ames, R. S.,
Chambers, J.,
Ellis, C.,
Elshourbagy, N.,
Foley, J. J.,
Schmidt, D. B.,
Muccitelli, R. M.,
Jenkins, O.,
Murdock, P. R.,
Herrity, N. C.,
Halsey, W.,
Sathe, G.,
Muir, A. I.,
Nuthulaganti, P.,
Dytko, G. M.,
Buckley, P. T.,
Wilson, S.,
Bergsma, D. J.,
and Hay, D. W. P.
(1999)
Mol. Pharmacol.
56,
657-663 |
7. | Stadel, J. M., Wilson, S., and Bergsma, D. J. (1997) Trends Pharmacol. Sci. 18, 430-437[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Bergsma, D. J.,
Eder, C.,
Gross, M.,
Kersten, H.,
Sylvester, D.,
Appelbaum, E.,
Cusimano, D.,
Livi, G. P.,
McLaughlin, M. M.,
and Kasyan, K.
(1991)
J. Biol. Chem.
266,
23204-23214 |
9. | Aiyar, N., Baker, E., Wu, H. L., Nambi, P., Edwards, R. M., Trill, J. J., Ellis, C., and Bergsma, D. J. (1994) Mol. Cell. Biochem. 131, 75-86[Medline] [Order article via Infotrieve] |
10. | Miyazaki, J., Araki, K., Yamato, E., Ikegami, H., Asano, T., Shibasaki, Y., Oka, Y., and Yamamura, K. (1990) Endocrinology 127, 126-132[Abstract] |
11. | Goetz, A. S., Andrews, J. L., Littleton, T. R., and Ignar, D. M. (2000) J. Biomol. Screening 5, 377-384[Medline] [Order article via Infotrieve] |
12. | Rorsman, P., and Trube, G. (1986) J. Physiol. (Lond.) 374, 531-550[Abstract] |
13. | Briscoe, C. P., Hanif, S., Arch, J. R., and Tadayyon, M. (2001) Cytokine 14, 225-229[CrossRef][Medline] [Order article via Infotrieve] |
14. | Ignar, D. M., and Rees, S. (2000) in Handbook of Experimental Pharmacology (Kenokin, J. , and Angus, J. A., eds), Vol. 148 , Springer-Verlag, Berlin |
15. |
Bokoch, G. M.,
Katada, T.,
Northup, J. K.,
Hewlett, E. L.,
and Gilman, A. G.
(1983)
J. Biol. Chem.
258,
2072-2075 |
16. |
Hordijk, P. L.,
Verlaan, I.,
Vancorven, E. J.,
and Moolenaar, W. H.
(1994)
J. Biol. Chem.
269,
645-651 |
17. | Coleman, D. L. (1978) Diabetologia 14, 141-148[Medline] [Order article via Infotrieve] |
18. | Gottlicher, M., Widmark, E., Li, Q., and Gustafsson, J. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4653-4657[Abstract] |
19. | Cistola, D. P., Hamilton, J. A., Jackson, D., and Small, D. M. (1988) Biochemistry 27, 1881-1888[Medline] [Order article via Infotrieve] |
20. | Tietz, N. W. (1996) in Tietz Fundamentals of Clinical Chemistry (Burtis, C. A. , and Ashwood, E. R., eds) , p. 789, WB Saunders, Philadelphia |
21. | Lee, K. C., Chou, K. J., Cheng, J. S., Wang, J. L., Tang, K. Y., Tseng, L. L., and Jan, C. R. (2001) Pharmacol. Toxicol. 88, 20-26[Medline] [Order article via Infotrieve] |
22. |
Stein, D. T.,
Stevenson, B. E.,
Chester, M. W.,
Basit, M.,
Daniels, M. B.,
Turley, S. D.,
and McGarry, J. D.
(1997)
J. Clin. Invest.
100,
398-403 |
23. |
Gravena, C.,
Mathias, P. C.,
and Ashcroft, S. J. H.
(2002)
J. Endocrinol.
173,
73-80 |
24. |
Dobbins, R. L.,
Chester, M. W.,
Stevenson, B. E.,
Daniels, M. B.,
Stein, D. T.,
and McGarry, J. D.
(1998)
J. Clin. Invest.
101,
2370-2376 |
25. | Dobbins, R. L., Chester, M. M., Daniels, M. B., McGarry, J. D., and Stein, D. T. (1998) Diabetes 47, 1613-1618[Abstract] |
26. | Sato, Y., and Henquin, J. C. (1998) Diabetes 47, 1713-1721[Abstract] |
27. | Komatsu, M., Yajima, H., Yamada, S., Kaneko, T., Sato, Y., Yamauchi, K., Hashizume, K., and Aizawa, T. (1999) Diabetes 48, 1543-1549[Abstract] |
28. | Prentki, M., Tornheim, K., and Corkey, B. E. (1997) Diabetologia 40, S32-S41[CrossRef][Medline] [Order article via Infotrieve] |
29. | Moore, S. A. (1993) Adv. Exp. Med. Biol. 331, 229-233[Medline] [Order article via Infotrieve] |
30. |
Yaney, G. C.,
Korchak, H. M.,
and Corkey, B. E.
(2000)
Endocrinology
141,
1989-1998 |
31. | Weng, L., Davies, M., and Ashcroft, S. J. H. (1993) Cell. Signal. 5, 777-786[CrossRef][Medline] [Order article via Infotrieve] |
32. | Gregersen, S., and Ahren, B. (1996) Pancreas 12, 48-57[Medline] [Order article via Infotrieve] |
33. | Carpentier, A., Mittelman, S. D., Bergman, R. N., Giacca, A., and Lewis, G. F. (2000) Diabetes 49, 399-408[Abstract] |
34. | Sako, Y., and Grill, V. E. (1990) Endocrinology 127, 1580-1589[Abstract] |
35. | Eitel, K., Staiger, H., Brandhorst, D., Brendel, M. D., Bretzel, R. G., Haering, H. U., and Kellerer, M. (2002) Diabetologia 45, A149 |
36. | Tokuyama, Y., Sturis, J., Depaoli, A. M., Takeda, J., Stoffel, M., Tang, J. P., Sun, X. H., Polonsky, K. S., and Bell, G. I. (1995) Diabetes 44, 1447-1457[Abstract] |
37. |
Lee, Y.,
Hirose, H.,
Ohneda, M.,
Johnson, J. H.,
Mcgarry, J. D.,
and Unger, R. H.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10878-10882 |
38. |
Svennerholm, L.
(1968)
J. Lipid Res.
9,
570-579 |
39. | Salem, N., Jr., Litman, B., Kim, H. Y., and Gawrisch, K. (2001) Lipids 36, 945-959[Medline] [Order article via Infotrieve] |
40. |
Kim, H. Y.,
Akbar, M.,
Lau, A.,
and Edsall, L.
(2000)
J. Biol. Chem.
275,
35215-35223 |
41. | Mattson, M. P., Duan, W., Pedersen, W. A., and Culmsee, C. (2001) Apoptosis 6, 69-81[CrossRef][Medline] [Order article via Infotrieve] |
42. | Kim, H. Y., Edsall, L., Garcia, M., and Zhang, H. (1999) Adv. Exp. Med. Biol. 447, 75-85[Medline] [Order article via Infotrieve] |