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Dopamine-Mediated Autocrine Inhibitory Circuit Regulating Human Insulin Secretion in Vitro

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We describe a negative feedback autocrine regulatory circuit for glucose-stimulated insulin secretion in purified human islets in vitro. Using chronoamperometry and in vitro glucose-stimulated insulin secretion measurements, evidence is provided that dopamine (DA), which is loaded into insulin-containing secretory granules by vesicular monoamine transporter type 2 in human β-cells, is released in response to glucose stimulation. DA then acts as a negative regulator of insulin secretion via its action on D2R, which are also expressed on β-cells. We found that antagonism of receptors participating in islet DA signaling generally drive increased glucose-stimulated insulin secretion. These in vitro observations may represent correlates of the in vivo metabolic changes associated with the use of atypical antipsychotics, such as increased adiposity.
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Dopamine-Mediated Autocrine Inhibitory Circuit
Regulating Human Insulin Secretion in Vitro
Norman Simpson,* Antonella Maffei,* Matthew Freeby, Steven Burroughs,
Zachary Freyberg, Jonathan Javitch, Rudolph L. Leibel, and Paul E. Harris
Division of Endocrinology (N.S., M.F., S.B., P.E.H.) and Center for Molecular Recognition (Z.F., J.J.),
Columbia University Medical College, and The Naomi Berrie Diabetes Center (M.F., R.L.L., P.E.H.), New
York, New York 10032; and The Institute of Genetics and Biophysics A. Buzzati-Traverso (A.M.),
Consiglio Nazionale delle Ricerche, 80125 Naples, Italy
We describe a negative feedback autocrine regulatory circuit for glucose-stimulated insulin secretion
in purified human islets in vitro. Using chronoamperometry and in vitro glucose-stimulated insulin
secretion measurements, evidence is provided that dopamine (DA), which is loaded into insulin-
containing secretory granules by vesicular monoamine transporter type 2 in human
-cells, is released
in response to glucose stimulation. DA then acts as a negative regulator of insulin secretion via its
action on D2R, which are also expressed on
-cells. We found that antagonism of receptors partici-
pating in islet DA signaling generally drive increased glucose-stimulated insulin secretion. These in
vitro observations may represent correlates of the in vivo metabolic changes associated with the use
of atypical antipsychotics, such as increased adiposity. (Molecular Endocrinology 26: 0000 –0000, 2012)
The
-cells of the islets of Langerhans integrate a vari-
ety of external signals into the timely release of phys-
iologically appropriate amounts of insulin and thus accu-
rately regulate blood glucose levels commensurate with
metabolic demand. Some external signals act as amplify-
ing agents that have little or no effect by themselves but
enhance the sensitivity of the
-cell glucose-sensing appa-
ratus (reviewed in Ref. 1). For example, certain amino
acids synergize with D-glucose in promoting insulin secre-
tion by
-cells. Net insulin production and glucose ho-
meostasis is regulated by other small molecules as well,
including several classical neurotransmitters (2, 3) that
act directly on
-cells and indirectly through other tissues
active in glucose homeostasis such as liver and skeletal
muscle. Neurotransmitters participating in glucose ho-
meostasis can be released from sympathetic and parasym-
pathetic innervation, the adrenal medulla, or as we dem-
onstrate in this report, directly from islets acting in an
autocrine or paracrine manner to regulate islet insulin
secretion.
Comparative microanatomy of human vs. rodent islets
and islet innervation reveals important differences that
may impact operant mechanisms of glucose homeostasis
(4). Relative to the structure of mouse islets, human islets
are sparsely innervated with few contacts to autonomic
and cholinergic axons (5). Moreover, in human islets,
sympathetic axons are associated with the smooth muscle
cells of blood vessels located around and deep within the
islet rather than directly contacting
-cells. To reconcile
the apparent autonomy of human islets with the known
effects of autonomic stimulation on rodent islet hormone
secretion, it has been suggested that neurotransmitter
spillover from innervation might be responsible for
downstream effects on hormone secretion (6). However,
an alternate possibility is autocrine and/or paracrine re-
lease of insulin secretory modulators.
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2012 by The Endocrine Society
doi: 10.1210/me.2012-1101 Received March 19, 2012. Accepted July 13, 2012.
* N.S. and A.M. have contributed equally to this report.
Abbreviations: AMPT,
-Methylparatyrosine; ATA, atypical antipsychotic drugs; BBB,
blood-brain barrier; BTCP, benzothiophenylcyclohexylpiperidine; CNS, central nervous
system; DA, dopamine; DAT, DA transporter; L-DOPA, L-3,4-dihydroxyphenylalanine; D2R,
DA type 2 receptor; dpm, decays per minute; DTBZ, dihydrotetrabenazine; GSIS, glucose-
stimulated insulin secretion; 5-HT, serotonin; IPGTT, intraperitoneal glucose tolerance
testing; KRBB, Krebs Ringers bicarbonate buffer; MAT, monoamine transporter; nCFM,
Nafion-coated carbon fiber microelectrodes; SD, Sprague Dawley; TBZ, tetrabenazine;
T2D, type 2 diabetes; VMAT2, vesicular monoamine type 2 transporter; ZF, Zucker fatty.
ORIGINAL RESEARCH
Mol Endocrinol, October 2012, 26(10):00000000 mend.endojournals.org 1
Molecular Endocrinology. First published ahead of print August 21, 2012 as doi:10.1210/me.2012-1101
Copyright (C) 2012 by The Endocrine Society
Negative feedback regulation and paracrine or auto-
crine signaling are common control mechanisms within
the central nervous system (CNS). For example, in mam-
malian brain, the nigrostriatal dopamine (DA) system is
necessary for voluntary motor activity. It is well estab-
lished that the activity of striatal neurons is regulated by
autoregulatory negative feedback loops (reviewed in Ref.
7) where released DA acts on presynaptic DA type 2 re-
ceptors (D2R) to decrease DA synthesis and release (8),
thereby reducing downstream signaling to postsynaptic
neurons.
As in the CNS, gene expression studies reveal that hu-
man islet tissue expresses a variety of molecules associ-
ated with the biosynthesis, storage, degradation, and re-
sponse to several neurotransmitters (9), including DA
(10).
-Cells express vesicular monoamine type 2 trans-
porters (VMAT2) (11), a molecule critical for the vesicu-
lar storage of DA (12), and DA type 1–5 receptors (13),
and DA is present in rodent
-cell vesicles (14). In this
report, we show evidence that DA is stored within human
pancreatic islets, released in response to glucose stimula-
tion, and acts on D2R (also expressed by human
-cells)
resulting in the down-regulation of insulin secretion. The
existence of a DA-mediated negative feedback regulatory
circuit in human islets may be particularly relevant in the
context of the association between the use of atypical
antipsychotic drugs (ATA) and development of metabolic
syndrome and type 2 diabetes (T2D). Given that the single
unifying property of ATA is their D2R antagonist activ-
ity, the prediction is that D2R blockade would blunt the
endogenous DA- and D2R-mediated negative feedback in
glucose-stimulated insulin secretion (GSIS), and we pro-
vide evidence that this is indeed the case in human islets.
Materials and Methods
Drugs and reagents
GBR 12909 dihydrochloride (vanoxerine), benzothiophenylcyclo-
hexylpiperidine (BTCP),
-methylparatyrosine (AMPT), haloperidol
hydrochloride, serotonin (5-HT), sulpiride, DA hydrochloride, quin-
pirole hydrochloride, clozapine, and D-glucose were obtained from
Sigma-Aldrich Corp. (St. Louis, MO). Tetrabenazine (TBZ) was ob-
tained from Tocris Bioscience (Ellisville, MO). Dihydrotetrabenazine
(DTBZ) was obtained from the National Institute of Mental Health’s
Chemical Synthesis and Drug Supply Program. Olanzapine was ob-
tained from E. Lilly (Indianapolis, IN). [ring 2,5,5-
3
H]DA was ob-
tained from American Radiolabeled Chemicals (St. Louis, MO). All
other chemicals were of the highest commercial quality available.
Pancreas and islet procurement and islet culture
Whole human pancreata from donors without known his-
tory of diabetes and fixed in 10% neutral buffered formalin
were procured from the National Disease Research Interchange
(Philadelphia, PA). Human islets isolated from cadaveric non-
diabetic donors were obtained from the Integrated Islet Distri-
bution Program (City of Hope National Medical Center, Du-
arte, CA). The average purity of islets was 90 5% (SEM)as
determined by dithizone staining, the average age of the donors
(n 36) was 42 2yr(
SEM). The average body mass index was
32 1(
SEM). The isolated human islets were normally cultured
in supplemented CMRL-1066 medium for no longer than 2 d
before being shipped. On arrival, islets were placed in CMRL-
1066 medium containing 5.5 mMglucose, 10% fetal bovine
serum, 100 U/ml penicillin, and 100
g/ml streptomycin and
incubated at 37 C with 5% CO
2
. All cell culture media and
supplements were obtained from Life Technologies (Grand Is-
land, NY). Tissue culture plates were obtained from Falconware
(Becton-Dickinson, Inc., Oxnard, CA). Islets used in these anal-
yses were cultured for at least 24 h but for no longer than 5 d. All
experiments regarding human islets and pancreata were ap-
proved by our Institutional Review Board.
Immunohistochemistry and
colocalization microscopy
Fixed pancreas tissue was embedded in paraffin and 5-
m
sections on glass slides prepared. Tissue was initially evaluated
by hematoxylin and eosin staining; if autolysis was present,
specimens were not evaluated in the study. Next, sections were
stained for both insulin and D2R. The primary antibodies used,
guinea pig antiinsulin (A0564; Dako, Carpinteria, CA) and a
rabbit polyclonal anti-D2R (PA1-23584; Thermo Scientific,
Rockford, IL), were chosen based on previous experience and
published research (15). Primary antibodies were incubated on
paraffin-embedded slides overnight at 4 C with the exception of
insulin. Insulin was incubated for 2 h at room temperature.
Secondary antibodies conjugated to fluorescein isothiocyanate
or Cy5 were diluted to 1:100 (Jackson ImmunoResearch, West
Grove, PA). All slides were coverslipped with Vectashield 4,6-
diamidino-2-phenylindole mounting medium (Vector Labora-
tories, Burlingame, CA), stored in the dark at 4 C, and analyzed
within1dofstaining. Slides were evaluated on a Nikon Eclipse
80i microscope using QCapture 51 to image insulin and D2R
immunofluorescent colocalization.
Analysis of D2R, VMAT2, DAT, and LAT1
transcripts by RT-PCR
Human pancreas (catalog item 540023-41) and striatum
(catalog item 540135-41) total RNA were from Agilent Tech-
nologies (MVP human normal adult tissue total RNA; Santa
Clara, CA). Total RNA from islets or purified acinar tissue (9)
was isolated using the RNA RNeasy Mini Kit (QIAGEN, Va-
lencia, CA). cDNA was generated using the VILO cDNA syn-
thesis kit (Life Technologies). All PCR assays were performed
using the amount of cDNA obtained retro-transcribing 30 ng
total RNA. Platinum PCR SuperMix High Fidelity (Life Tech-
nologies) was used for semiquantitative PCR assays at the con-
ditions recommended by the manufacturer; in particular, 40
cycles of PCR amplification were performed with an annealing
temperature of 56 C and an extension time of 45 sec. Accumu-
lation of specific transcripts was measured by real-time PCR,
using the SmartCycler System (Cepheid, Sunnyvale, CA). The
QuantiTect SYBR Green PCR Kit (QIAGEN) was used to per-
form all the quantitative PCR assays with annealing tempera-
2Simpson et al. Autocrine Neuroregulation of Insulin Secretion Mol Endocrinol, October 2012, 26(10):0000 0000
tures of 55–60 C and extension times of 30 45 sec, depending
on the couple of primers used. All the primers (Supplemental
Table 1, published on The Endocrine Society’s Journals Online
web site at http://mend.endojournals.org) were synthesized by
Eurofins MWG Operon (Huntsville, AL), except for those spe-
cific for human
-actin (QuantiTect Primer Assay; QIAGEN).
Quantitative RT-PCR reagent controls (reagents without any
template or with 30 ng non-retro-transcribed RNA) were in-
cluded in all the assays. Each assay was run in triplicate and
independently repeated at least three times to verify the results;
the mean copy number was used for analysis. The relative
amount of specific transcripts was calculated by the compara-
tive cycle threshold method given by Schmittgen and Livak (16).
To correct for sample to sample variations in quantitative RT-
PCR efficiency and errors in sample quantitation, the level of
ACTB transcripts was tested for use in normalization of specific
RNA levels.
Experimental animals
All animal studies were reviewed and approved by the Insti-
tutional Animal Care and Use Committee at Columbia Univer-
sity’s College of Physicians and Surgeons. Male Sprague-Dawley
(SD) and Zucker obese rats were obtained from Harlan Labo-
ratories (Somerville, NJ). Rodents were housed under condi-
tions of controlled humidity (55 5%), temperature (23 1
C), and lighting (lights on 0600–1800 h) with ad libitum access
to standard laboratory rat chow and water.
Intraperitoneal glucose tolerance testing (IPGTT)
IPGTT was performed as previously described in 6-h-fasted
unanesthetized rats as described previously (17). Sixty minutes
before IPGTT, isoflurane anesthesia of male rats was induced
and maintained in an oxygen mixture. The anesthetized rats
were administered TBZ, GBR 12909, or vehicle alone at the
indicated dose by iv injection at the penile vein. The animals
were fully recovered for at least 30 min before receiving IPGTT,
which was performed without anesthesia.
Insulin, DA, and DNA assays
Human insulin measurements were performed using the Alph-
aLISA human insulin research immunoassay (kit AL2904Cm;
PerkinElmer, Waltham MA). Rat Insulin measurements were per-
formed using ELISA (kit DOP31-K01; Eagle Bioscience,
Nashua, NH). DA measurements were performed using ELISA
(kit BA E-6300; Rocky Mountain Diagnostics, Colorado Springs,
CO). All measurements were made following the kit manufac-
turer’s protocols using a BioTek Synergy 2 reader with the
appropriate filters (BioTek, Winooski, VT) DNA concentra-
tion measurements, used to normalize the mass of islets used
in the static incubation experiments, were performed using
the QuantiFluor double-stranded DNA system (catalog item
E2670; Promega Corp., Madison, WI) according to manufac-
turer’s recommendations.
Human islet insulin secretion and DA uptake
assays
For the static incubation experiments, human islets were
washed once in Krebs Ringers bicarbonate buffer (KRBB) (125
mMNaCl, 4.8 mMKCl, 2.6 mMCaCl
2
, 1.2 mMMgCl
2
,25mM
NaHCO
3
,10mMHEPES (final pH 7.4) supplemented with
0.2% (wt/vol) serum albumin, preheated to 37 C, and bubbled
with 95% O
2
and 5% CO
2
] and then preincubated for1hin
KRBB with 1.5 mMglucose (basal conditions).
In some experiments, this preincubation step was performed
in the presence of 250 mg/liter AMPT. Islets were washed again
in KRBB and seeded into 48-well tissue culture plates (BD Fal-
con 353230; BD Biosciences, Bedford, MA) at 100–500 islets/
ml 0.5 ml/well in KRBB. Solutions containing the indicated
drug and/or glucose were prepared at twice the indicated con-
centration in KRBB (e.g. 30 mMglucose plus 100 nMDTBZ).
These prewarmed (37 C) solutions (0.5 ml) were then added to
wells with islets to yield the indicated final concentrations. Islets
were then cultured for1hat37Cwith an atmosphere of 5%
CO
2
/95% Air. At the end of each incubation period, a 0.5-ml
aliquot of the culture medium was carefully collected to avoid
aspiration of islets and frozen (80 C) for analysis of insulin
concentration and the remaining 0.5 ml set aside and frozen
(80 C) for later DNA measurements. The insulin concentra-
tions measurements were normalized to the DNA content of the
well to compensate for the variability of the well-seeding
technique.
For the perfusion experiments, eight to 18 islets (diameters
200
m) were perfused in a temperature- and pH-controlled
100-
l chamber slide (Ibidi
slide I luer, 0.4 mm height; Auto-
Mate Scientific, Inc., Berkeley, CA USA) and fractions collected
for later analysis of insulin concentration. The perfusion appa-
ratus consisted of 1) a temperature- and pH-controlled reservoir
containing KRBB with constant aeration, connected by C-Flex
tubing to 2) a peristaltic pump (dual-channel microperfusion
pump; AutoMate Scientific), followed by 3) an inline solution
heater and 4) the Ibidi chamber slide. The Ibidi chamber slide
was mounted on a temperature-controlled transparent glass
stage (TC-invivo; Bioscience Tools, San Diego, CA). Feedback
control of the chamber temperature and perfusate was achieved
with an inline temperature probe connected to a temperature
controller (TC-TP; Bioscience Tools). The line out of the Ibidi
chamber was covered with a 50-
m nylon mesh to prevent
escape of islets, and the 100-
l chamber was partially filled with
inert polystyrene beads (160–200
m diameter; Solohill Engi-
neering Inc., Ann Arbor, MI) to reduce chamber dead volume
and aid in the enumeration and measurement of islets and their
diameters. The chamber line out was connected by 0.031-in.
tubing to a Gilson fraction collector.
Islets in KRBB were loaded into the Ibidi chambers, and
the contents of the transparent chambers were examined and
the number of islets and their size recorded. The filled cham-
bers were placed in the apparatus and perfused at a rate of
600–700
l/min with KRBB with 1.5 mMglucose for 30 min
to equilibrate. At time zero, the collection of 0.5-min frac-
tions began. Twenty minutes later, the glucose concentration
was raised to 15 mMglucose in the presence and absence of
TBZ and an additional 30 min of islet perfusion was per-
formed. Collected fractions were stored at 80 C for later
assay of insulin content by AlphaLISA. The average coeffi-
cient of variation for AlphaLISA-based insulin measurements
was less than 12%. At the end of the experiment, the chamber
was reexamined to confirm the islet count. In some experi-
ments, where DA and insulin concentration measurements
were both measured in the same fraction, we increased the
number of islets per chamber to 1200 200 to allow simul-
taneous detection by ELISA.
Mol Endocrinol, October 2012, 26(10):0000 0000 mend.endojournals.org 3
Measurements of tritiated DA uptake by islets were per-
formed as follows: islets were seeded (about 500 per well) into
BD Falcon cell culture inserts for 24-well plates (8.0-
m pores,
translucent polyethylene terephthalate membrane) containing
0.5 ml KRBB with 1.5 or 8.0 mMglucose and 0.5% wt/wt BSA.
To some wells, haloperidol was added to a final concentration
of 10
M. Islets were next incubated at 37 C in an atmosphere of
5% CO
2
/95% Air for 45 min. Next, some wells received GBR
12909 and/or BCTP to a final concentration of 10
M. Islets were
incubated for 30 min followed by the addition of 1 10
7
decays
per minute (dpm)/well of [
3
H]DA. Islet cultures were incubated for
1 h and followed by washing and harvesting. Islets within the
inserts were washed three times with 1 ml cold 4 C Ringers with
0.5% BSA using gentle suction to not disrupt the islets. After the
third wash, 250
l 0.1 NHCl was added to the insert and the
contents frozen at 80 C until analysis. Upon thawing, 25
l
supernatant was removed for DNA assay and the remainder mixed
with 5 ml Ecolite scintillation cocktail. Samples were counted in a
calibrated liquid scintillation counter, and the background-cor-
rected disintegrations per minute for each sample was calculated
and then normalized to the well’s DNA content.
Human islet glucose-stimulated DA release assays
Nafion-coated carbon fiber microelectrodes (nCFM) (diam-
eter 30
mlength 100
m) were obtained from World Pre-
cision Instruments, Sarasota, FL (CFN30-100) or coated de
novo as previously described (18). Chronoamperometric and
voltammetry measurements were performed with a MicroC po-
tentiostat (World Precision Instruments) using a combined
reference/auxiliary electrode. The reference electrode was a dry-
type Ag/AgCl cylinder (World Precision Instruments), encapsu-
lated in an insulating tube, as supplied with the MicroC such
that the active surface was a 2-mm-diameter Ag/AgCl disc. The
time constant on the MicroC was set at 1 msec rise time. To
drive the potentiostat, we used a Rigol DG1000 series function/
arbitrary waveform generator (Rigol USA, Oakwood Village,
OH). The MicroC potentiostat converts detected currents to a
voltage signal that was collected using a 100-msec sampling
period via a high-resolution USB data logger (ADC-20; Pico
Technology, Cambridgeshire, UK) and the supplied PicoLog
data acquisition software. The data signal was low-pass filtered
(50 Hz) to remove all noise at frequencies above 50 Hz from
the signal before sampling and then stored in a personal com-
puter in real time. Data reduction was performed using Excel
software (Microsoft Corp., Redmond, WA) and macros created
with the visual basic editor.
Before using the electrodes to measure DA release by islets in
vitro, we characterized the in vitro response of the nCFM to
both authentic DA and 5-HT. The nCFM were first precondi-
tioned in PBS by applying a 70-Hz triangle wave (from 0 to 3.0
V relative to the Ag/AgCl reference electrode) for 20 sec, fol-
lowed by holding the electrode in PBS at 1.5 V for 20 sec (19),
followed by holding the electrode in 150 mMNaCl (pH 9.5) at
1.2 V for 5 min. Cyclic voltammograms for KRBB alone, KRBB
with authentic DA added, and KRBB with authentic 5-HT
added were collected using the following parameters: wave-
form, triangular; period, 8 sec; applied voltage maximum, 1000
mV; applied voltage minimum, 200 mV; scan rate, 350 V/sec,
and duration, more than 12 cycles. The potentiostat gain used
was 1 nA/mV to 100 pA/mV (see Supplemental Information).
In preliminary experiments, we found that using our instru-
mentation, the application of cyclic voltammetry to the mea-
surement of DA release was insensitive to DA concentrations
below 100 nM. Instead, we applied a related previously de-
scribed square-wave or normal-pulse voltammetry/chronoam-
perometry technique (20–22). After the activation of the nCMF
electrodes described above, square-wave pulses cor-
responding to the oxidation peak of DA were ap-
plied to the detection of DA released from islets in
vitro. The parameters used were as follows: wave-
form, square; duty cycle, 50%; applied voltage
maximum, 220 mV; applied voltage minimum,
80 mV; and period, 8 sec. The potentiostat gain was
set a 1 pA/mV. The nCFM and reference electrode
were placed in the well of a 24-well tray filled with
1.0 ml Ringers buffer and the background current
allowed to stabilize for 10 min under an applied
voltage of 220 mV, after which the average back-
ground current was set to 0. The working electrode
was situated 1–2 mm above the well floor. The tray
and its contents were maintained at 37 C in a tem-
perature-monitored micro-incubator. Next, the
square waveform was applied to KRBB in the well,
and the current vs. time profile generated by the
applied voltage profile was collected for 20 min. No
current was observed in KRBB supplemented with
up to 20 mMglucose in the absence of added islets.
During this time, human islets were washed in
warm (37 C) KRBB and suspended at 1000 islets/ml
in 0.5 ml KRBB. At time zero, the data collection
was reinitiated, and 0.5 ml KRBB was removed
from the well and replaced with the 0.5-ml suspen-
sion of human islets in KRBB. At 15 min, the glu-
cose concentration was raised to 1.5 mMwith a
FIG. 1. Human D2R and insulin staining in a representative pancreas section and
islet from a healthy nondiabetic subject. Left panels, Immunofluorescent staining of
a representative islet from a nondiabetic subject for D2R (top,green), insulin (middle,
red), merged with 4,6-diamidino-2-phenylindole staining (bottom); right panel,
wide-field view of section showing islet and distribution of D2R and insulin staining.
D2R staining was confined mainly to the insulin-positive islet.
4Simpson et al. Autocrine Neuroregulation of Insulin Secretion Mol Endocrinol, October 2012, 26(10):0000 0000
concentrated solution of glucose prepared in
Ringers, at 30 min, the glucose concentration in
the well was again raised to 15 mMglucose. At
1 h, data acquisition was stopped, and two
thirds of culture supernatant was carefully re-
moved to not disturb the islets and replaced with
fresh warm KRBB four times, resulting in a di-
lution of glucose to a final concentration of less
than 0.2 mM. Data acquisition was started again,
and after 20 min, the glucose concentration was
raised to 30 mMglucose. At 120 min, the poten-
tiostat gain was reduced to 10 pA/mV from 1
pA/mV, and the islet-containing well was spiked
with authentic DA to a final concentration of
500 nM. About 8 min later, an additional spike
of authentic DA was added to bring the final
concentration to 800 nMDA.
Statistical evaluation
Data are presented as the mean SEM. The
significance of comparisons of statistical means
were calculated using the two-tailed Students ttest.
Statistically significant insulin pulse pulses were
identified with the Cluster Analysis program, a
computerized pulse analysis algorithm (23). Test
cluster sizes for the nadir and peak widths were
assigned to 4. The minimum tstatistics was speci-
fied to 4.0 for both upstrokes and downstrokes.
The settings detected peaks with less than 1%
false-positive errors. Cluster analyses also pro-
vided information regarding peak height and peri-
odicity. A sliding two-tailed Student’s ttest with a
three-point window was also used to compare
peak heights between DTBZ-treated and un-
treated islet perfusion profiles.
Results
D2R colocalize with insulin staining in
human pancreas sections and are
found predominantly in islets
Rubí et al. (13) showed colocalization of
insulin and D2R in purified human islets. In
sections of human pancreas, we examined
the distribution of D2R and its relationship
to insulin staining of islets in situ (Fig. 1).
The staining pattern of D2R and its overlap
with insulin staining is similar to that of
VMAT2 (15) and suggests that D2R is pres-
ent in insulin granules on resting
-cells.
The fluorescent immunohistochemical la-
beling of sections also showed that within
the pancreas, most of the D2R expression
was limited to
-cells.
D2R are highly expressed in the stria-
tum, on dopaminergic presynaptic neurons
FIG. 2. Expression of D2R transcripts in human striatum, pancreas, and purified
cadaveric islets. A and B, RT-PCR semiquantitative assay using the primer pairs
4hD2_F/3hD2_R (A) and 4hD2_F/4hD2_R (B), amplifying cDNA derived from both
the long and short mRNA isoform of D2R. The 234-bp and 532-bp products
expected from the amplification of D2RS cDNA are not visible in A and B at 40
cycles. When the reaction in A was extended to 55 cycles, the D2RS cDNA 234-bp
produce became visible. C, RT-PCR semiquantitative assay using the primer pair
Long_hD2_F/Long_hD2_R, specific for the long isoform of D2R. D, RT-PCR
semiquantitative assay using the primer pair Short_hD2_F/3hD2R, specific for the
short isoform of D2R. The 215-bp product expected from the amplification of D2RS
cDNA is not visible. When the reaction in D was extended to 55 cycles, the D2RS
cDNA 215-bp product became visible. E, RT-PCR semiquantitative assay using the
primer pair 2hD2_F/2hD2R, amplifying cDNA derived from all the known mRNA
isoforms of D2R. F, RT-PCR semiquantitative assay using the primer pair
Hs_ACTB_2_SG, amplifying
-actin-specific cDNA. This figure shows the results of
one of five similar experiments. All primer pairs and primer sequences are listed in
Supplemental Table 1.
Mol Endocrinol, October 2012, 26(10):0000 0000 mend.endojournals.org 5
as well as on medium spiny neurons and corticostriatal
neurons. Alternative splicing of the D2R gene results in at
least two transcript variants encoding different isoforms:
the abundant long isoform (D2RL) including 29 amino
acids derived from exon 6 and a short isoform (D2RS)
missing these amino acids (24). To further understand the
function of D2R in
-cells, we examined the expression of
D2R at the transcript level in human total pancreas and
purified cadaveric islets (Fig. 2). Using primers specific for
both long and short isoforms, short isoform alone, long
isoform alone, and total D2R (Supplemental Table 1), we
amplified cDNA prepared from pancreas, purified islets,
and as a control, human striatum. We found that, similar
to striatum, D2RL was the predominantly
expressed D2R isoform in human islets
with D2RS not visible under the experi-
mental conditions. Furthermore, the sum of
the transcripts derived from all the known
isoforms of D2R mRNA was higher in islets
relative to total pancreas. The amount of
D2R message in human islets was compa-
rable to that found in human striatum.
We used real-time PCR with the same set
of primers to precisely quantitate levels of
specific D2R isoforms (Fig. 3) in purified
human islet, total human pancreas, and hu-
man striatum cDNA. We found that 1) in
human islets, the abundance of D2RL and
D2RS transcripts is approximately 5-fold
greater than that found in human striatum
and 50 times higher than that found in total
pancreas; 2) the amount of D2RL-specific
mRNA paralleled the amount of total D2R
transcripts; and 3) the copy number of D2RS-
specific mRNA, although detectable, was low
and consistent with the semiquantitative results
shown in Fig. 2 (data not shown).
In a separate set of experiments, we
probed cDNA, prepared from whole pan-
creas, purified islets, and purified acinar
exocrine tissue, for the expression of insulin
(INS), as an islet purity control, and four
other products of the SLC (solute carrier)
gene superfamily, SLC7A5 (also known as
LAT1 or CD98 light chain), SLC3A2 (also
known as MDU1 or CD98), SLC18A2
(also known as VMAT2), and SLC6A3
(also known as DA reuptake transporter,
DAT) relevant to a putative DA-mediated
negative feedback regulatory circuit of in-
sulin secretion. As expected, both insulin
and VMAT2 gene expression was more
than 30-fold enriched in islets relative to whole pancreas
and more than 4 10
3
-fold greater than the level mea-
sured in purified exocrine tissue. We also found that islets
were enriched, relative to whole pancreas or pancreas
exocrine tissue, for the expression of SLC7A5,SLC3A2,
and SLC6A3.
Antagonism of monoamine transporters (MAT)
and DA receptors enhances insulin secretion by
human islets in vitro
The transport of monoamine neurotransmitters (e.g.
DA) across cellular and vesicular membranes is mediated
by members of a family of transporters known as MAT.
FIG. 3. Expression of D2R, insulin (INS), SLC7A5,SLC3A2,SLC18A2, and SLC6A3
mRNA in purified human cadaveric islets by quantitative RT-PCR. cDNA was amplified
with the primer pair 2hD2_F/2hD2R, amplifying all D2R-specific cDNA (D2R), the primer
pair Long_hD2_F/Long_hD2_R specific for the long isoform of D2R (D2RL). The amounts
of transcripts in each tissue were normalized to ACTB transcript content in the
respective samples and the results for D2R are reported as relative to the amount of
specific cDNA found in striatum, which was arbitrarily set to 1. The results for insulin,
SLC7A5,SLC3A2,SLC18A2, and SLC6A3 are reported as relative to the amount of
specific cDNA found in whole pancreas, which was arbitrarily set to 1. Error bars show
the SEM. The differences in expression of D2R between striatum, pancreas, and islets
were significant at the P0.05 level using Student’s ttest. *, Significant difference
from whole pancreas or exocrine tissue at the P0.05 level using Student’s ttest.
6Simpson et al. Autocrine Neuroregulation of Insulin Secretion Mol Endocrinol, October 2012, 26(10):0000 0000
In a previous report, we demonstrated that TBZ-medi-
ated antagonism of VMAT2 resulted in enhanced GSIS of
isolated islets (25). To determine whether a similar effect
might be observed in cultures of human islets, we exam-
ined GSIS in the presence and absence of DTBZ (Fig. 4).
Because DTBZ effects in this context are believed to be
mediated through DA depletion (26, 27), we also tested
GSIS in the presence of DA. As an additional control, we
tested the effects of haloperidol, a first-generation anti-
psychotic and antagonist of DA action at D2R. We found
that DTBZ enhanced GSIS at both high (15 mM) and low
(8 mM) glucose concentrations but did not stimulate in-
sulin secretion in the absence of glucose (Fig. 4). The
amount of glucose-stimulated insulin release, normalized
to islet DNA content in the sample, in these static incubation
assays was similar to that detected previously (28). As reported
for rodent islets (29), we found that DA (1.0
M) significantly
inhibited GSIS, whereas the antagonist of DA action at D2R,
haloperidol, enhanced GSIS. Additional studies of GSIS using
islets from different donors confirmed the generality of our
findings with DTBZ, DA, haloperidol, and the tyrosine hy-
droxylase inhibitor AMPT (Fig. 5). As additional controls, we
tested the effects of quinpirole (a selective D2 receptor agonist)
and sulpiride (a selective D2 and D3 receptor antagonist) on
human islet insulin secretion. As expected, agonism and antag-
onism at D2R, respectively, decreased and increased GSIS in
human islets (Fig. 5).
In the CNS behind the blood-brain barrier (BBB), DA
released at the synapse is recycled into vesicles by the
action of the DAT. Here, DAT inhibitors increase ex-
tracellular DA concentrations because diffusion of hy-
drophilic molecules like DA is restricted by the BBB
(30), but inactivation of DAT by knockout reduces
tissue DA content (31). Because islets are not bordered
by a BBB, yet are heavily vascularized and richly per-
fused, we hypothesized that inhibition of DAT might
affect a net loss of DA signaling and inhibition of DAT
by GBR 12909 might have similar effects on GSIS as
did DA depletion by DTBZ. We found that GBR 12909
enhanced GSIS in human islets in vitro (Fig. 5), perhaps
by blocking recycling and allowing DA to diffuse out of
the cultured islet tissue. Supporting this idea was the
additional finding that islet tissue stimulated with 8 mM
glucose and treated with GBR 12909 contained less DA
than control islet tissue stimulated with 8 mMglucose
alone (Fig. 6A).
On the basis of these results, we formed the working
hypothesis that DA stored in
-cell vesicles by action of
VMAT2 is coreleased into the extracellular space with
insulin upon glucose stimulation. DA acting at D2R in an
autocrine manner or on nearby adjacent
-cells partially
suppresses insulin secretion. Remaining DA molecules
not internalized with D2R, or removed by the circulation,
would be recycled via DAT.
FIG. 4. Measurement of GSIS in islets from a single donor in the presence of glucose, TBZ, haloperidol, or DA. The islets were incubated in basal
KRBB for 1 h and then seeded in wells and exposed to the indicated glucose and drug concentration for 1 h. The mean insulin concentration,
normalized to the well DNA content, was then determined for sextuplicate wells. Error bars indicate the SEM. *, Significantly different (P0.0.05)
from the wells with 8 mMglucose alone; **, significantly different from wells with 15 mMglucose alone; ***, significantly different from wells
with 3 mMglucose alone.
Mol Endocrinol, October 2012, 26(10):0000 0000 mend.endojournals.org 7
Antagonism of VMAT2 by TBZ enhances insulin
secretion in human islets in vitro by increasing
secretion pulse height
To better understand how this DA circuit might be
operating in islets, we examined the kinetics of GSIS un-
der conditions of VMAT2 antagonism by DTBZ. The
insulin concentration vs. time profile arising from perfu-
sion of human islets with 1.5 mMglucose vs. 15 mMglu-
cose with and without added DTBZ was measured and
revealed insulin pulses (Fig. 7). After the perfusate glucose
concentration was increased to 15 mM
from 1.5 mM, the insulin concentration
in collected fractions increased above
basal levels. At 15 mMglucose, several
significant peaks of insulin secretion
(averaging about 7 10
2
amol/ml per
islet) were observed with an average
period of 5 0.5 min (SEM), consistent
with previous reports (32, 33). In the
presence of DTBZ, the amplitudes of
the insulin pulses were significantly in-
creased without a significant change in
their periodicity. The mass of insulin
secreted, as determined by the area un-
der the curve during the high-glucose
perfusion, was higher in the presence of
DTBZ than in its absence, consistent
with the static incubation experiments
(Fig. 5).
Human islets in vitro release DA in
a glucose-dependent manner
In investigating a possible
-cell au-
tocrine or paracrine circuit involving
DA-mediated regulation of GSIS and
having shown that human islets and
-cells express D2R, and respond to
DA and DA depletion in vitro, we next
examined whether islets also released
DA in response to glucose stimulation.
Amperometry and voltammetry at mi-
croelectrodes have been demonstrated
to possess sufficient sensitivity and
temporal resolution to detect exocyto-
sis of DA in the CNS (34). Similar tech-
niques have been successfully applied
to measurements of 5-HT and acetyl-
choline exocytosis from rodent and hu-
man islets (6, 35–37), but release of DA
has not been characterized. To demon-
strate release of DA, we first character-
ized the response of our detection sys-
tem to DA and 5-HT (see Supplemental
Fig. 1). Under our conditions, the nCFM was at least
50-fold more responsive to DA than to 5-HT.
Next, we probed the supernatant of islet cultures stim-
ulated with glucose using an electrochemical technique
and nCFM with preferential sensitivity to DA. The read-
out of this chronoamperometric technique is a current
proportional to the DA concentration (Fig. 8C). Islets
were first incubated in KRBB without glucose, followed
by KRBB supplemented with glucose to 1.5 mMand then
FIG. 5. Measurement of GSIS in islets from multiple donors in the presence of glucose, TBZ,
GBR 12909, DA, AMPT (pretreatment), 5-HT, and selected D2R agonists and antagonists.
Static incubations were performed as indicated in Fig. 4. Each islet donor was tested against
1.5 mMglucose, 15 mMglucose, and one or more of the indicated compounds. Each bar
represents the mean values obtained from three or more islet donors. For each donor and
condition tested, the GSIS (nanograms insulin per nanogram DNA) was normalized to mean
GSIS obtained in 1.5 mMglucose for that donor. Error bars indicate the SEM. The mean GSIS
for each compound tested was then compared with mean GSIS obtained in 15 mMglucose
alone. *, Significantly different (P0.0.05) from the wells with 15 mMglucose alone;
**, significantly different (P0.0.05) from the wells with 1.5 mMglucose alone.
8Simpson et al. Autocrine Neuroregulation of Insulin Secretion Mol Endocrinol, October 2012, 26(10):0000 0000
to 15 mMglucose. The average currents detected under
these conditions were 21 2, 23 3, and 64 3pA
(mean SEM), respectively. Although the observed av-
erage current at 15 mMglucose was significantly
greater (P0.001) than the current at no glucose and
1.5 mMglucose, the current detected at 1.5 mMglucose
was not significantly different from the current ob-
served at no glucose, although there was evidence of an
increased number of current spikes, which may signify
evidence of pulsatile DA release. After incubation in 15
mMglucose, the glucose concentration was reduced
nearly 30-fold to 0.5 mM, and we observed a corre-
sponding drop in the average current. Subsequently, we
increased the glucose concentration to 30 mMglucose
and observed a rise in the current. The average current
rose significantly above the baseline, but this time, we
also observed decay in the average current, possibly
signaling DA depletion in the cultured islets. To pro-
vide evidence that the electrodes were still responsive to
DA, at the end of the experiments, we added known
concentrations of authentic exogenous DA to the islet
cultures and measured the faradaic currents. On this
basis, we estimate that DA release by islets was about
10
2
fmol/islet per current spike during stimulation by
FIG. 6. A, DA content of islet tissue and supernatants after GSIS. Static incubations were performed with 500 islets per well in KRBB with the
indicated concentration of DTBZ or GBR 12909, with islets placed within the well inserts. Islets were obtained from two or more different donors
as indicated by the value of n. At the end of the incubation, the supernatants were harvested and the DA and insulin content assayed. Islet tissue
contained within the inserts were washed twice and then harvested in 1 ml buffer and assayed for insulin, DA, and DNA. DA and insulin
concentration measurements were normalized to the DNA content of the well, and the mean value of at least triplicate wells was calculated. To
compare groups, the amount of DA measured in the supernatant of each donor after stimulation with 8 mMglucose was set to represent 100%.
All measurements in subsequent groups (supernatant vs. intracellular vs. treatment with GBR 12909) from that donor were expressed as the
fraction of 8 mMglucose control supernatant value. B, DA release during perfusion experiments. Insulin and DA content were measured in
selected fractions collected (flow rate 750
l/min at one fraction/30 sec) from approximately 1200 islets perfused in KRBB buffer with 8.0 mM
glucose. Results are from a representative experiment in a series of two. *, Significant difference at the P0.05 level. C, DA uptake by cultured
human islets. Islets were first cultured in 10
Mhaloperidol to block [
3
H]DA binding to D2R. To these cultures 10
MGBR 12909 or BCTP was then
added to inhibit DAT function. [
3
H]DA was then added to the cultures for 1 h followed by washing and harvesting the islets. The amount of DA
associated with the islets was then determined by scintillation counting, and the results are given as dpm normalized to the well DNA content.
Each bar represents the average results obtained from two different islet donors, and the uptake for each condition was determined as the mean
of triplicate wells. Error bars represent the SEM. The results are given as background-corrected normalized dpm or as a percentage of control using
the mean dpm value from cultures with 1.5 mMglucose and no haloperidol or DAT inhibitors added as the denominator. *, Significantly different
(P0.0.05) from the wells with 1.5 mMglucose alone; **, significantly different (P0.0.05) from the wells containing 1.5 mMglucose and 10
Mhaloperidol.
Mol Endocrinol, October 2012, 26(10):0000 0000 mend.endojournals.org 9
15 mMglucose and similar to release of 5-HT as re-
ported previously for mouse pancreatic
-cells (36).
To confirm the findings of DA release by human islets
in response to glucose stimulation, we measured the DA
concentration in supernatants obtained from the static
incubation experiments outlined above using the tech-
nique of ELISA. Here DA-specific antibodies are used to
capture and detect DA from solution. The cross-reactivity
of this assay with other endogenous monoamines (e.g.
norepinephrine) was less than 1%. We found the amount
of DA released by islets, normalized to the DNA content
of the well, was significantly greater in 15 mMglucose
relative to 1.5 mMglucose (mean concentration 0.28
0.03 vs. 0.61 0.07 ng DA/ng DNA) (Fig. 6A). We next
measured both the DA and insulin content of the collected
fraction from islet perfusion experiments per-
formed under stimulation by 8.0 mMglucose
(Fig. 6B). We found that peaks of DA immu-
noreactivity were largely, but not entirely, co-
incident with peaks of insulin immunoreactiv-
ity. Lastly, we measured tritiated DA uptake
by cultured human islets. Total islet uptake of
DA under the conditions tested was likely to
reflect both binding and internalization by
D2R and a component specifically mediated
by DAT. To discriminate between these com-
ponents, total uptake was measured in Ringers
buffer with 1.5 mMglucose and the presence
and absence of a D2R antagonist (haloperidol)
and one or both of two specific DAT inhibitors
[GBR 12909 (38) or BTCP (39)] (Fig. 6C). It
was observed that addition of GBR 12909
and/or BCTP reduced the islet associated ra-
dioactivity to about 60% of the control level.
Haloperidol blocked up to 30% of the total
uptake of [
3
H] DA. When GBR 12909 or
BCTP was added to the cultures containing
haloperidol, the internalized counts dropped
an additional 35% to about 45% of the total
control radioactivity and were significantly
(P0.05) less than internalized counts ob-
served in the presence of haloperidol blocking
alone. Similar results were found in cultures
performed in 8.0 mMglucose.
Antagonism of VMAT2 or DAT reduces
glucose excursions during IPGTT in the
Zucker obese T2D rat model in vivo
Our in vitro experiments suggested that the
monoamine transporters, DAT and VMAT2,
were important control points in the regula-
tion of GSIS. To determine whether this obser-
vation could be extended to in vivo models, we
next examined the in vivo effects of TBZ and GBR 12909
in the Zucker fatty (ZF) rodent model of human pre-type
2 diabetes (Fig. 9, top and middle panel). As an additional
control, we examined the effects of GBR 12909 on blood
glucose levels during IPGTT in SD rats (Fig. 9, middle
panel). In vivo inhibition of both VMAT2 and DAT dur-
ing IPGTT reduced the glucose excursions after ip glucose
challenge. The reduced glucose excursions measured after
GBR 12909 administration and 30 min after glucose chal-
lenge were accompanied by increased serum insulin levels
in both the SD and ZF rodent models (Fig. 9, bottom
panel). Previously, we reported similar effects on insulin
secretion when TBZ was administered before in vivo glu-
cose challenge (i.e. increased serum insulin) (25).
FIG. 7. Effects of raising glucose from 1.5 to 15 mMin the presence and absence of
DTBZ on the release of insulin from perfused human islets from two different
donors. Upper panel shows results of 18 islets (donor a); bottom panel shows results
of eight islets (donor b). Human islets were perfused (700
l/min) in KRBB under
basal glucose conditions for 20 min followed by raising the perfusate glucose
concentration to 15 mMglucose with and without added DTBZ. Insulin
concentrations were measured in duplicate or triplicate from 30-sec fractions of the
perfusate. Cluster analysis (23) of the insulin concentration vs. time profile revealed
the significant peaks of insulin secretion (indicated by asterisks). **, Peaks obtained
during perfusion in 15 mMglucose with DTBZ that were significantly larger ( P
0.05) than those obtained without DTBZ as determined by a sliding ttest. The area
under the curve was determined using the trapezoidal rule. Two representative
experiments from a series of three are shown.
10 Simpson et al. Autocrine Neuroregulation of Insulin Secretion Mol Endocrinol, October 2012, 26(10):0000 0000
ATA enhance in vitro insulin secretion by
human islets
The ATA (e.g. clozapine and olanzapine), are charac-
terized by potent antagonist activity at DA D2 and D3
and 5-HT2A receptors. In immunohistochemistry exper-
iments, we demonstrated that D2R colocalized with insu-
lin on
-cells and that D2RL was the
predominant isoform expressed by
-cells. In static incubation experi-
ments, we demonstrated that human
islet GSIS was inhibited by 5-HT, DA
(1
M), and the D2R selective agonist
quinpirole and enhanced by D2R an-
tagonists such as haloperidol and
sulpiride. On this basis, we predicted
that ATA might also enhance human
islet GSIS. We performed static incuba-
tion measurements of GSIS in the pres-
ence of a range of concentrations of
either clozapine or olanzapine using is-
lets obtained from several donors (Figs.
10 and 11). In general, we found that
both clozapine and olanzapine signifi-
cantly enhanced GSIS but that the ef-
fect differed in magnitude from donor
to donor (Fig. 11).
Discussion
The regulatory role of intrinsic inner-
vation on islet insulin secretion has
been expertly summarized by Ahrén
(3) and Thorens (40). Although past
studies have mostly relied on rodent is-
lets, more recent microanatomical
studies in human islets suggest a dimin-
ished role for direct innervation of
-cells in regulation of insulin secretion
(5). Our present study provides evi-
dence of a novel DA-mediated regula-
tory autocrine circuit for insulin secre-
tion by human islets in vitro. Such a
circuit may partially support overall
regulation of GSIS given the observed
relative autonomy of human islets
from sympathetic innervation (5).
An appreciation of the presence of
monoamine neurotransmitters within
islets and the role of these transmitters
in regulation of glucose secretion be-
gan in the 1960s (41). During the fol-
lowing five decades, it became clear not
only that rodent islets contain neurotransmitters (42) but
also that
-cell vesicles contained insulin and 5-HT (43,
44) and/or DA as well. Next, it was found that
-cells
probably express most if not all the enzymes needed for de
novo synthesis of DA, its catabolism (13, 45, 46), and the
FIG. 8. Chronoamperometry of glucose-sensitive islet DA release. A, Input voltage waveform
to the potentiostat (square wave pulses 0.08 to 0.220 V vs. Ag/AgCl with an 8-sec period
and 50% duty cycle). B, Output current signals from a nCFM in KRBB. The initial voltage pulse
was accompanied by a large increase in the double-layer current, which discharged well
before the end of 50% duty cycle mark. C, The faradaic current (i.e. generated from the
oxidation of DA) was measured during the last second of the 220-mV pulse (gray bar in A
and B). The glucose concentration was varied as indicated, and the time vs. current data were
plotted (C) and the average currents at each glucose concentration calculated. At the end of
the experiment (time 2 h) authentic DA was added to the well to obtain the corresponding
faradaic current to confirm sensitivity to DA. Results are shown from a representative
experiment in a series of three.
Mol Endocrinol, October 2012, 26(10):0000 0000 mend.endojournals.org 11
VMAT that sequester cytosolic neurotransmitters into se-
cretory storage granules (11). In the human pancreas, the
expression of VMAT2 is largely confined to
-cell gran-
ules containing insulin (11, 15). Low levels of VMAT2
expression are present in exocrine pancreas (47) and en-
docrine PP cells, but
-cells (glucagon) and
-cells (soma-
tostatin) do not stain for VMAT2 protein (15). Similar to
VMAT2,
-cells also express D2R, and as shown in this
report, D2R expression in the pancreas is also largely
confined to the
-cells. We found that in man, similar to
what was observed in the rat (13), islets predominantly
express the D2RL-specific isoform with D2RS-specific
transcript representing a minority. In the CNS, neuronal
DA release can be down-regulated using DA receptor ago-
nists via DA autoreceptors (8). Knockout experiments in
mice reveal that this autoreceptor’s activity is most closely
associated with the D2RS isoform (48). Additional study
will be needed to understand the molecular physiology of
islet DA receptor activity.
Based upon our earlier experiments, we hypothesized
that DA, accumulated in
-cell vesicles by action of
VMAT2, is released into the extracellular space along
with insulin upon glucose stimulation. DA acting at D2R,
FIG. 9. Top panel, TBZ reduces the blood glucose excursion during
Intraperitoneal Glucose Tolerance Test (IPGTT) in male ZF (obese)
rats. Mean blood glucose values during IPGTT of ZF rats treated
with vehicle alone (f,n4) or with TBZ at 2.25
g/g body weight
(,n5). Error bars represent the SEM. The area under the curve
(AUC) for each rodent’s blood glucose vs. time profile was
calculated using the trapezoid rule. The average AUC was
calculated and compared between control and TBZ-treated groups.
The Pvalue of the significance of the comparison of AUC by ttest is
shown. Middle panel, GBR 12909 reduces the blood glucose
excursions during IPGTT in male ZF (obese) and SD rats. Mean blood
glucose values during IPGTT of rats treated with vehicle alone (Œ
and F,n5) or with GBR 12909 at 5.0
g/g body weight (and
E,n5). The comparison of AUC was performed as detailed
above. Bottom panel, Sera collected at 30 min after IPGTT from the
experiments shown above were assayed for insulin. The mean
serum insulin concentrations for treated rats were compared with
the values of untreated rats. The significance of comparison was
determined by ttest and is indicated within the bar.
FIG. 10. Measurement of GSIS in islets from a single donor in the
presence of the ATA olanzapine. The islets were incubated in basal
KRBB for 1 h and then seeded in wells and exposed to the indicated
glucose and drug concentration for 1 h. The mean insulin
concentration, normalized to the well DNA content, was then
determined for sextuplicate wells. Mean insulin concentrations,
normalized to the well DNA content, under different conditions were
compared with the mean response to 15 mMglucose alone by ttest.
The significance of each comparison, where P0.05, is shown. Error
bars indicate the SEM.
12 Simpson et al. Autocrine Neuroregulation of Insulin Secretion Mol Endocrinol, October 2012, 26(10):0000 0000
newly exposed at the cell surface after a round of GSIS or
expressed on vicinal
-cells, partially suppresses insulin
secretion and residual DA, not internalized with D2R or
removed by the circulation, would then be recycled by the
action of DAT (49). To test this hypothesis, we first ex-
amined the expression of DAT in the pancreas. We ob-
served that DAT was preferentially expressed in the islet
tissue relative to whole pancreas (6-fold) and at least
10-fold greater than that measured in exocrine tissue. We
next studied the in vitro effects of TBZ’s VMAT2 antag-
onism as well as its DA-depleting action on GSIS. ELISA-
based measurements revealed that intra-islet DA content
was reduced and GSIS was enhanced in the presence of
DTBZ. In addition, we found that inhibition of L-3,4-
dihydroxyphenylalanine (L-DOPA, the direct precursor
of DA) synthesis by AMPT, an inhibitor of tyrosine hy-
droxylase, also enhanced islet GSIS. In this regard, it is
interesting to note that islets also express the large neutral
amino acid transporter system, composed of a heavy
subunit encoded the SLC3A2 gene and a light subunit encoded
by the SLC7A5 gene. This heterodimer preferentially trans-
ports branched-chain and aromatic (tryptophan and tyrosine)
amino acids, including L-DOPA (50), and is highly expressed in
brain capillary endothelial cells that form the BBB (51). As a
consequence of the expression of DAT, the large
neutral amino acid transporter system and
VMAT2, islet
-cells appear to have the capacity to
remove DA and its biosynthetic precursors (e.g.
L-DOPA) (52) from the circulation and concen-
trate DA intravesicularly. As a direct consequence
of expression of these molecules by dopaminergic
neurons, the concentration of DA at release sites
have been estimated to be about 1–100
M, well
within the upper limits of DA concentrations tested
here. Nevertheless, it is not excluded that other in-
travesicular monoamines (e.g. norepinephrine) are
active here. In addition to blocking DA transport at
the level of insulin granules, we also tested the ef-
fects of antagonism of the
-cell’s plasma mem-
brane DAT. As in the case of VMAT2 inhibition,
we found that antagonism of DAT by GBR 12909
enhanced islet GSIS. We speculated that inhibition
of DAT in islets might result in loss of DA signal-
ing, and although our in vitro and in vivo experi-
ments support this conclusion, additional study
and attention to possible off-target effects is
needed.
We also found that human islet GSIS was
enhanced in the presence of antagonists of
D2R such as sulpiride and haloperidol. D2R
agonists such as exogenous DA and quin-
pirole, as expected, diminished human islet
GSIS. A study of the kinetics of islet GSIS in the presence
of DTBZ revealed that enhanced insulin secretion was
probably due to increases in the amplitude of pulsatile
release rather than increases in the periodicity of insulin
release. Lastly, we probed human islets in vitro for DA
uptake mediated by DAT and glucose-dependent DA re-
lease. We found that islets could internalize exogenous
[
3
H]DA through a GBR 12909- or BTCP-sensitive path-
way. We also observed that the amount of DA released
into culture supernatants increased with increasing ambi-
ent glucose concentrations and that DA release was con-
comitant with insulin release. The confirmation of glu-
cose-dependent DA release by human islets and the
additional above observations satisfy the conditions re-
quired by a DA-mediated autocrine feed-forward nega-
tive regulatory circuit.
We next examined whether this putative in vitro circuit
could be manipulated to improve glucose clearance in
vivo using a pre-type 2 diabetes rodent model. We found
that antagonism of VMAT2 reduced the in vivo glucose
excursions after glucose tolerance testing and that inhibi-
tion of DAT by GBR 12909 resulted in enhanced serum
insulin concentrations after an ip glucose challenge as
might be expected based on the results of our in vitro
FIG. 11. Measurement of GSIS in islets from multiple donors treated with
olanzapine or clozapine. Static incubations were performed as indicated in Fig. 4.
Each islet donor was tested against 15 mMglucose alone and 15 mMglucose plus
the indicated compounds and dose. For each donor and condition tested, the GSIS
(nanograms insulin per nanogram DNA) was normalized to mean GSIS obtained in
15 mMglucose alone for that donor to obtain the relative change. The mean relative
change of GSIS among all donors tested for each compound tested (black bar) was
then compared with mean GSIS obtained in 15 mMglucose alone. The Pvalue
obtained by Student’s ttest, where P0.05, is shown for each condition tested in
the population.
Mol Endocrinol, October 2012, 26(10):0000 0000 mend.endojournals.org 13
experiments, although it is not excluded that these drugs
target sites beyond the endocrine pancreas. Although
these results suggest that off-label use of existing VMAT2
inhibitors or D2R antagonists might be applied to en-
hance insulin secretion in response to glucose; in practice,
however, placing too high a demand on insulin secretion
could result in
-cell exhaustion (53). Consistent with
this, the too-much-of-a-good-thing hypothesis (54) was
proposed, in part, based on the results of the ADOPT
study (55) showing that driving increased insulin secre-
tion with sulfonylureas had worse long-term outcomes
than pharmacotherapy with insulin sensitizers.
Because ATA have potent D2R antagonist activity
(56), they have potential to act in a D2R-dependent man-
ner at targets found within the islet autocrine DA circuit.
In addition, the use of ATA has been associated with
increased risk of developing T2D. In the past, increased
risk of developing T2D associated with ATA use has been
ascribed to increases in adiposity (and consequent insulin
resistance) mediated via their CNS effects on food intake
and physical activity (57, 58). We reasoned that an addi-
tional contributor to overall risk might be that, if certain
ATA directly antagonize D2R on
-cells, an additional
increase in insulin secretion might result through the DA/
D2R-mediated autocrine feed-forward mechanism pro-
posed above. We tested the effects of ATA clozapine and
olanzapine on human islet GSIS in vitro and found that
GSIS in the presence of these ATA was indeed enhanced.
Similar experiments measuring GSIS in rodent islets
treated with ATA have shown mixed results, ranging
from no effect to effects only on basal insulin secretion
(59 61), suggesting additional study is needed.
Because both drugs also have 5-HT receptor antago-
nist activity (including at 5-HT2A receptors), we also
tested the effects of 5-HT on in vitro GSIS. As previously
reported for rodent islets (62), we found that 5-HT inhib-
ited islet GSIS, and thus it is reasonable to conclude that
5-HT receptor antagonism would parallel the effects of
D2R antagonism. Thus, ATA via antagonism at D2R and
5-HT receptors could potentially promote
-cell loss by
allowing
-cells to produce more insulin (63, 64) with
resultant endoplasmic reticulum stress (65). Lastly, the
efficacy of bromocriptine, a potent D2R agonist, in the
management of T2D (66) might also be explained via its
local effects on
-cell insulin secretion in addition to its
central effects.
Although human
-cells may express the full comple-
ment of enzymes needed for de novo synthesis of DA,
-cells are probably not the only source of DA in the
pancreas. Other DA sources might include, for example,
spillover from the nerve terminals that innervate the pan-
creas (5, 67), cells of the exocrine pancreas, and/or re-
uptake from arterial blood, because the pancreas is fed by
three interlocking arterial circles (47). Reliance on such
sources of spillover DA would require that
-cells express
DAT. In this report, we have shown indirect evidence that
that DAT is expressed in islets and has a role in regulating
GSIS in vitro and in vivo.
In this context, it is interesting to note that the brain is
only a minor source of peripheral DA. As is the case for
5-HT, the major source of circulating DA is the gastroin-
testinal tract including the gastric mucosa (68). This fact,
coupled with the findings that there are large postprandial
spikes of serum DA in man (52) and that in vitro islets
have a functional DAT, suggests that DA in its role of
regulating
-cell insulin secretion represents an anti-in-
cretin hypothesized to explain the beneficial effects of
bariatric surgery on T2D (69). Here surgical procedures
that possibly lower circulating DA levels (e.g. sleeve gas-
trectomy with duodenal switch) might be predicted to
have enhanced insulin secretion. Driving increased
-cell
function, particularly when there is weight loss failure,
could result in the eventual postoperative reoccurrence of
T2D as has been observed in a subpopulation of bariatric
surgery patients (70).
Acknowledgments
Address all correspondence and requests for reprints to: Paul E.
Harris, Ph.D., Division of Endocrinology, Department of Med-
icine, Columbia University Medical College, 650 West 168th
Street, BB 2006, New York, New York 10032. E-mail:
peh1@columbia.edu.
This work was supported by the U.S. Public Health Service,
National Institutes of Health, National Institute of Diabetes and
Digestive and Kidney Diseases (5 R01 DK063567) and the Helms-
ley Charitable Trust (09PG-T1D020) (http://helmsleytrust.org).
The funders had no role in study design, data collection and anal-
ysis, decision to publish, or preparation of the manuscript.
Disclosure Summary: P.E.H. and A.M. are inventors on U.S.
patent applications 20100204258 and 20110118300. N.S.,
M.F., S.B., Z.F., J.J., and R.L.L. have nothing to declare.
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16 Simpson et al. Autocrine Neuroregulation of Insulin Secretion Mol Endocrinol, October 2012, 26(10):0000 0000
... Dopamine (DA) is a neurotransmitter in the brain, and alterations in DA metabolism are related to the pathophysiology of neuropsychiatric disorders, such as Parkinson's disease, schizophrenia, and some developmental disorders. In addition to its role in the brain, DA also plays pivotal physiological functions in peripheral tissues, such as natriuresis in the kidney (1-3), glucose-stimulated insulin secretion in the pancreas (4)(5)(6)(7), liquid clearance in the lung (8), and acid secretion in the stomach (9,10). DA regulates these functions via DA receptors belonging to G protein-coupled receptors. ...
... DA in the adult pancreas has a physiological function to suppress glucose-stimulated insulin secretion. DA in β-cells is reported to be synthesized from extracellular DOPA by AADC (4,36). However, a recent study has revealed differential TH expression among mice strains, that is, 35-fold more THpositive β-cells in islets of PWK/PhJ and CAST/EiJ mice than of C57BL/6J mice (37). ...
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... In particular, the most expressed subtypes are D2 (long and short) and D3. A subtle inhibitory paracrine and autocrine function on β-cells has been postulated for local dopamine sourced from dietary precursors (e.g., L-DOPA) and/or noradrenergic fibers [31][32][33]. In fact, rodent and human islets express enzymes such as tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase (AADC), vesicular monoamine transporter 2 (VMAT2) and dopamine active transporter (DAT) to produce and store dopamine [31]. ...
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... With these premises, a physiological autocrine/paracrine function of dopamine for a fine regulation of β-cells insulin secretion has also been proposed [14]. Dopamine can be produced locally by its precursors originating from the diet, stored in insulin vesicles via vesicular monoamine transporter-2 (VMAT-2) and then secreted together with insulin [16,17]. β-cells also express transporters for dopamine (DAT) and its amino-acids precursors (LAT1 and 2) [15,18]. ...
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... The role of dopamine in insulin secretion has repeatedly been considered for decades [44]. Dopamine was detected inside β cells [45,46] and in insulin secretory granules [47], from where it can be co-secreted with insulin. Inhibitory effects of exogenous dopamine on insulin secretion from islets were reported in 2005 [48]. ...
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A variety of behavioral disorders are associated with addictions, impulsivity, obsessive-compulsive behaviors, and some personality disorders, which are collectively known as reward deficiency syndrome. These disorders are also associated with diverse digestive disorders. The enteric nervous system, a complex subdivision of the peripheral nervous system, and the central nervous system secrete diverse neurotransmitters including acetylcholine, dopamine, and serotonin. These neurotransmitters can have impaired functional competence due to digestive dysfunction. Hypothalamic-gut axis plays a vital role in nutrient selection. Therapeutic targets to prevent food addiction and neuroendocrine-mediated obesity stem from prodopamine regulation. Altered dopamine reward circuits are an antecedent to genetically induced survival panic, stress intolerance, pathologic food consumption, increased fat synthesis and storage, and digestive disorders. In contrast to current methods that utilize dopamine antagonistic or very powerful agonistic therapies and are based on the commonality of neural mechanisms between drug and glucose addiction, evidence from neuroimaging epigenetic studies strongly indicates that gentle dopamine agonist strategies targeting disrupted dopamine pathways make a significant contribution to achieving dopamine homeostasis and remitting digestive distress and dysbiosis. As a result, precision dopamine agonistic therapies for chronic addiction depend on scientifically sound and appropriate evidence-based early genetic risk determinations leading to precision personalized care of the patient. Multifaceted systems biology-based therapies have been shown to rebalance the sequela of genetically mediated neurotransmitter transactions; restore competent genetically regulated feedback; rebalance neurochemical crosstalk; and optimize health.
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... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
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