dopamine-mediated autocrine inhibitory circuit regulating...

Dopamine-Mediated Autocrine Inhibitory CircuitRegulating 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.), NewYork, 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 secretionin purified human islets in vitro. Using chronoamperometry and in vitro glucose-stimulated insulinsecretion 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 releasedin response to glucose stimulation. DA then acts as a negative regulator of insulin secretion via itsaction 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 invitro observations may represent correlates of the in vivo metabolic changes associated with the useof atypical antipsychotics, such as increased adiposity. (Molecular Endocrinology 26: 1757–1772, 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 withmetabolic demand. Some external signals act as amplify-ing agents that have little or no effect by themselves butenhance the sensitivity of the �-cell glucose-sensing appa-ratus (reviewed in Ref. 1). For example, certain aminoacids 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) thatact directly on �-cells and indirectly through other tissuesactive in glucose homeostasis such as liver and skeletalmuscle. 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 insulinsecretion.

Comparative microanatomy of human vs. rodent isletsand islet innervation reveals important differences thatmay impact operant mechanisms of glucose homeostasis(4). Relative to the structure of mouse islets, human isletsare sparsely innervated with few contacts to autonomicand cholinergic axons (5). Moreover, in human islets,sympathetic axons are associated with the smooth musclecells of blood vessels located around and deep within theislet rather than directly contacting �-cells. To reconcilethe apparent autonomy of human islets with the knowneffects of autonomic stimulation on rodent islet hormonesecretion, it has been suggested that neurotransmitterspillover from innervation might be responsible fordownstream 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 1944-9917Printed in U.S.A.Copyright © 2012 by The Endocrine Societydoi: 10.1210/me.2012-1101 Received March 19, 2012. Accepted July 13, 2012.First Published Online August 21, 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 nervoussystem; 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 tolerancetesting; 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.

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Negative feedback regulation and paracrine or auto-crine signaling are common control mechanisms withinthe central nervous system (CNS). For example, in mam-malian brain, the nigrostriatal dopamine (DA) system isnecessary for voluntary motor activity. It is well estab-lished that the activity of striatal neurons is regulated byautoregulatory 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 postsynapticneurons.

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 thisreport, we show evidence that DA is stored within humanpancreatic 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. Theexistence of a DA-mediated negative feedback regulatorycircuit in human islets may be particularly relevant in thecontext of the association between the use of atypicalantipsychotic drugs (ATA) and development of metabolicsyndrome and type 2 diabetes (T2D). Given that the singleunifying property of ATA is their D2R antagonist activ-ity, the prediction is that D2R blockade would blunt theendogenous DA- and D2R-mediated negative feedback inglucose-stimulated insulin secretion (GSIS), and we pro-vide evidence that this is indeed the case in human islets.

Materials and Methods

Drugs and reagentsGBR12909dihydrochloride (vanoxerine),benzothiophenylcyclo-

hexylpiperidine (BTCP), �-methylparatyrosine (AMPT), haloperidolhydrochloride, serotonin (5-HT), sulpiride, DA hydrochloride, quin-pirole hydrochloride, clozapine, and D-glucose were obtained fromSigma-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’sChemical Synthesis and Drug Supply Program. Olanzapine was ob-tained from E. Lilly (Indianapolis, IN). [ring 2,5,5-3H]DA was ob-tained from American Radiolabeled Chemicals (St. Louis, MO). Allother chemicals were of the highest commercial quality available.

Pancreas and islet procurement and islet cultureWhole human pancreata from donors without known his-

tory of diabetes and fixed in 10% neutral buffered formalinwere 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) asdetermined by dithizone staining, the average age of the donors(n � 36) was 42 � 2 yr (SEM). The average body mass index was32 � 1 (SEM). The isolated human islets were normally culturedin supplemented CMRL-1066 medium for no longer than 2 dbefore being shipped. On arrival, islets were placed in CMRL-1066 medium containing 5.5 mM glucose, 10% fetal bovineserum, 100 U/ml penicillin, and 100 �g/ml streptomycin andincubated at 37 C with 5% CO2. All cell culture media andsupplements 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. Allexperiments regarding human islets and pancreata were ap-proved by our Institutional Review Board.

Immunohistochemistry andcolocalization microscopy

Fixed pancreas tissue was embedded in paraffin and 5-�msections on glass slides prepared. Tissue was initially evaluatedby hematoxylin and eosin staining; if autolysis was present,specimens were not evaluated in the study. Next, sections werestained for both insulin and D2R. The primary antibodies used,guinea pig antiinsulin (A0564; Dako, Carpinteria, CA) and arabbit polyclonal anti-D2R (PA1-23584; Thermo Scientific,Rockford, IL), were chosen based on previous experience andpublished research (15). Primary antibodies were incubated onparaffin-embedded slides overnight at 4 C with the exception ofinsulin. Insulin was incubated for 2 h at room temperature.Secondary antibodies conjugated to fluorescein isothiocyanateor Cy5 were diluted to 1:100 (Jackson ImmunoResearch, WestGrove, 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 analyzedwithin 1 d of staining. Slides were evaluated on a Nikon Eclipse80i microscope using QCapture 51 to image insulin and D2Rimmunofluorescent colocalization.

Analysis of D2R, VMAT2, DAT, and LAT1transcripts 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; SantaClara, 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 performedusing the amount of cDNA obtained retro-transcribing 30 ngtotal 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, 40cycles of PCR amplification were performed with an annealingtemperature 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). TheQuantiTect SYBR Green PCR Kit (QIAGEN) was used to per-form all the quantitative PCR assays with annealing tempera-

1758 Simpson et al. Autocrine Neuroregulation of Insulin Secretion Mol Endocrinol, October 2012, 26(10):1757–1772


tures of 55–60 C and extension times of 30–45 sec, dependingon the couple of primers used. All the primers (SupplementalTable 1, published on The Endocrine Society’s Journals Onlineweb site at http://mend.endojournals.org) were synthesized byEurofins MWG Operon (Huntsville, AL), except for those spe-cific for human �-actin (QuantiTect Primer Assay; QIAGEN).Quantitative RT-PCR reagent controls (reagents without anytemplate or with 30 ng non-retro-transcribed RNA) were in-cluded in all the assays. Each assay was run in triplicate andindependently repeated at least three times to verify the results;the mean copy number was used for analysis. The relativeamount 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 ofACTB transcripts was tested for use in normalization of specificRNA levels.

Experimental animalsAll 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 � 1C), and lighting (lights on 0600–1800 h) with ad libitum accessto 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 minutesbefore IPGTT, isoflurane anesthesia of male rats was inducedand maintained in an oxygen mixture. The anesthetized ratswere administered TBZ, GBR 12909, or vehicle alone at theindicated dose by iv injection at the penile vein. The animalswere fully recovered for at least 30 min before receiving IPGTT,which was performed without anesthesia.

Insulin, DA, and DNA assaysHuman 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 theappropriate filters (BioTek, Winooski, VT) DNA concentra-tion measurements, used to normalize the mass of islets usedin the static incubation experiments, were performed usingthe QuantiFluor double-stranded DNA system (catalog itemE2670; Promega Corp., Madison, WI) according to manufac-turer’s recommendations.

Human islet insulin secretion and DA uptakeassays

For the static incubation experiments, human islets werewashed once in Krebs Ringers bicarbonate buffer (KRBB) (125mM NaCl, 4.8 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 25 mM

NaHCO3, 10 mM HEPES (final pH 7.4) supplemented with

0.2% (wt/vol) serum albumin, preheated to 37 C, and bubbledwith 95% O2 and 5% CO2] and then preincubated for 1 h inKRBB with 1.5 mM glucose (basal conditions).

In some experiments, this preincubation step was performedin the presence of 250 mg/liter AMPT. Islets were washed againin 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 indicateddrug and/or glucose were prepared at twice the indicated con-centration in KRBB (e.g. 30 mM glucose plus 100 nM DTBZ).These prewarmed (37 C) solutions (0.5 ml) were then added towells with islets to yield the indicated final concentrations. Isletswere then cultured for 1 h at 37 C with an atmosphere of 5%CO2/95% Air. At the end of each incubation period, a 0.5-mlaliquot of the culture medium was carefully collected to avoidaspiration of islets and frozen (�80 C) for analysis of insulinconcentration 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 thewell to compensate for the variability of the well-seedingtechnique.

For the perfusion experiments, eight to 18 islets (diameters �200 �m) were perfused in a temperature- and pH-controlled100-�l chamber slide (Ibidi �slide I luer, 0.4 mm height; Auto-Mate Scientific, Inc., Berkeley, CA USA) and fractions collectedfor later analysis of insulin concentration. The perfusion appa-ratus consisted of 1) a temperature- and pH-controlled reservoircontaining KRBB with constant aeration, connected by C-Flextubing to 2) a peristaltic pump (dual-channel microperfusionpump; AutoMate Scientific), followed by 3) an inline solutionheater and 4) the Ibidi chamber slide. The Ibidi chamber slidewas mounted on a temperature-controlled transparent glassstage (TC-invivo; Bioscience Tools, San Diego, CA). Feedbackcontrol of the chamber temperature and perfusate was achievedwith an inline temperature probe connected to a temperaturecontroller (TC-TP; Bioscience Tools). The line out of the Ibidichamber was covered with a 50-�m nylon mesh to preventescape of islets, and the 100-�l chamber was partially filled withinert polystyrene beads (160–200 �m diameter; Solohill Engi-neering Inc., Ann Arbor, MI) to reduce chamber dead volumeand aid in the enumeration and measurement of islets and theirdiameters. 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, andthe contents of the transparent chambers were examined andthe number of islets and their size recorded. The filled cham-bers were placed in the apparatus and perfused at a rate of600 –700 �l/min with KRBB with 1.5 mM glucose for 30 minto equilibrate. At time zero, the collection of 0.5-min frac-tions began. Twenty minutes later, the glucose concentrationwas raised to 15 mM glucose in the presence and absence ofTBZ and an additional 30 min of islet perfusion was per-formed. Collected fractions were stored at �80 C for laterassay of insulin content by AlphaLISA. The average coeffi-cient of variation for AlphaLISA-based insulin measurementswas less than 12%. At the end of the experiment, the chamberwas reexamined to confirm the islet count. In some experi-ments, where DA and insulin concentration measurementswere both measured in the same fraction, we increased thenumber of islets per chamber to 1200 � 200 to allow simul-taneous detection by ELISA.



Measurements of tritiated DA uptake by islets were per-formed as follows: islets were seeded (about 500 per well) intoBD Falcon cell culture inserts for 24-well plates (8.0-�m pores,translucent polyethylene terephthalate membrane) containing0.5 ml KRBB with 1.5 or 8.0 mM glucose and 0.5% wt/wt BSA.To some wells, haloperidol was added to a final concentrationof 10 �M. Islets were next incubated at 37 C in an atmosphere of5% CO2/95% Air for 45 min. Next, some wells received GBR12909 and/or BCTP to a final concentration of 10 �M. Islets wereincubated for 30 min followed by the addition of 1 � 107 decaysper minute (dpm)/well of [3H]DA. Islet cultures were incubated for1 h and followed by washing and harvesting. Islets within theinserts were washed three times with 1 ml cold 4 C Ringers with0.5% BSA using gentle suction to not disrupt the islets. After thethird wash, 250 �l 0.1 N HCl was added to the insert and thecontents frozen at �80 C until analysis. Upon thawing, 25 �lsupernatant was removed for DNA assay and the remainder mixedwith 5 ml Ecolite scintillation cocktail. Samples were counted in acalibrated liquid scintillation counter, and the background-cor-rected disintegrations per minute for each sample was calculatedand then normalized to the well’s DNA content.

Human islet glucose-stimulated DA release assaysNafion-coated carbon fiber microelectrodes (nCFM) (diam-

eter 30 �m � length 100 �m) were obtained from World Pre-cision Instruments, Sarasota, FL (CFN30-100) or coated denovo as previously described (18). Chronoamperometric andvoltammetry measurements were performed with a MicroC po-tentiostat (World Precision Instruments) using a combinedreference/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 suchthat the active surface was a 2-mm-diameter Ag/AgCl disc. The

time constant on the MicroC was set at 1 msec rise time. Todrive the potentiostat, we used a Rigol DG1000 series function/arbitrary waveform generator (Rigol USA, Oakwood Village,OH). The MicroC potentiostat converts detected currents to avoltage signal that was collected using a 100-msec samplingperiod via a high-resolution USB data logger (ADC-20; PicoTechnology, Cambridgeshire, UK) and the supplied PicoLogdata acquisition software. The data signal was low-pass filtered(�50 Hz) to remove all noise at frequencies above 50 Hz fromthe signal before sampling and then stored in a personal com-puter in real time. Data reduction was performed using Excelsoftware (Microsoft Corp., Redmond, WA) and macros createdwith the visual basic editor.

Before using the electrodes to measure DA release by islets invitro, we characterized the in vitro response of the nCFM toboth authentic DA and 5-HT. The nCFM were first precondi-tioned in PBS by applying a 70-Hz triangle wave (from 0 to �3.0V 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 mM NaCl (pH 9.5) at1.2 V for 5 min. Cyclic voltammograms for KRBB alone, KRBBwith authentic DA added, and KRBB with authentic 5-HTadded were collected using the following parameters: wave-form, triangular; period, 8 sec; applied voltage maximum, 1000mV; applied voltage minimum, �200 mV; scan rate, 350 V/sec,and duration, more than 12 cycles. The potentiostat gain usedwas 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 concentrationsbelow 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 invitro. The parameters used were as follows: wave-form, square; duty cycle, 50%; applied voltagemaximum, �220 mV; applied voltage minimum, �80 mV; and period, 8 sec. The potentiostat gain wasset a 1 pA/mV. The nCFM and reference electrodewere placed in the well of a 24-well tray filled with1.0 ml Ringers buffer and the background currentallowed to stabilize for 10 min under an appliedvoltage of �220 mV, after which the average back-ground current was set to 0. The working electrodewas situated 1–2 mm above the well floor. The trayand its contents were maintained at 37 C in a tem-perature-monitored micro-incubator. Next, thesquare waveform was applied to KRBB in the well,and the current vs. time profile generated by theapplied voltage profile was collected for 20 min. Nocurrent was observed in KRBB supplemented withup to 20 mM glucose in the absence of added islets.During this time, human islets were washed inwarm (37 C) KRBB and suspended at 1000 islets/mlin 0.5 ml KRBB. At time zero, the data collectionwas reinitiated, and 0.5 ml KRBB was removedfrom 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 mM with a

FIG. 1. Human D2R and insulin staining in a representative pancreas section andislet from a healthy nondiabetic subject. Left panels, Immunofluorescent staining ofa 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.



concentrated solution of glucose prepared inRingers, at 30 min, the glucose concentration inthe well was again raised to 15 mM glucose. At1 h, data acquisition was stopped, and twothirds of culture supernatant was carefully re-moved to not disturb the islets and replaced withfresh warm KRBB four times, resulting in a di-lution of glucose to a final concentration of lessthan 0.2 mM. Data acquisition was started again,and after 20 min, the glucose concentration wasraised to 30 mM glucose. At 120 min, the poten-tiostat gain was reduced to 10 pA/mV from 1pA/mV, and the islet-containing well was spikedwith authentic DA to a final concentration of500 nM. About 8 min later, an additional spikeof authentic DA was added to bring the finalconcentration to 800 nM DA.

Statistical evaluationData are presented as the mean � SEM. The

significance of comparisons of statistical meanswere calculated using the two-tailed Students t test.Statistically significant insulin pulse pulses wereidentified with the Cluster Analysis program, acomputerized pulse analysis algorithm (23). Testcluster sizes for the nadir and peak widths wereassigned to 4. The minimum t statistics 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 t test with athree-point window was also used to comparepeak heights between DTBZ-treated and un-treated islet perfusion profiles.

Results

D2R colocalize with insulin staining inhuman pancreas sections and arefound predominantly in islets

Rubí et al. (13) showed colocalization ofinsulin and D2R in purified human islets. Insections of human pancreas, we examinedthe distribution of D2R and its relationshipto insulin staining of islets in situ (Fig. 1).The staining pattern of D2R and its overlapwith insulin staining is similar to that ofVMAT2 (15) and suggests that D2R is pres-ent in insulin granules on resting �-cells.The fluorescent immunohistochemical la-beling of sections also showed that withinthe pancreas, most of the D2R expressionwas 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 purifiedcadaveric islets. A and B, RT-PCR semiquantitative assay using the primer pairs4hD2_F/3hD2_R (A) and 4hD2_F/4hD2_R (B), amplifying cDNA derived from boththe long and short mRNA isoform of D2R. The 234-bp and 532-bp productsexpected from the amplification of D2RS cDNA are not visible in A and B at 40cycles. When the reaction in A was extended to 55 cycles, the D2RS cDNA 234-bpproduce became visible. C, RT-PCR semiquantitative assay using the primer pairLong_hD2_F/Long_hD2_R, specific for the long isoform of D2R. D, RT-PCRsemiquantitative assay using the primer pair Short_hD2_F/3hD2R, specific for theshort isoform of D2R. The 215-bp product expected from the amplification of D2RScDNA is not visible. When the reaction in D was extended to 55 cycles, the D2RScDNA 215-bp product became visible. E, RT-PCR semiquantitative assay using theprimer pair 2hD2_F/2hD2R, amplifying cDNA derived from all the known mRNAisoforms of D2R. F, RT-PCR semiquantitative assay using the primer pairHs_ACTB_2_SG, amplifying �-actin-specific cDNA. This figure shows the results ofone of five similar experiments. All primer pairs and primer sequences are listed inSupplemental Table 1.



as well as on medium spiny neurons and corticostriatalneurons. Alternative splicing of the D2R gene results in atleast two transcript variants encoding different isoforms:the abundant long isoform (D2RL) including 29 aminoacids derived from exon 6 and a short isoform (D2RS)missing these amino acids (24). To further understand thefunction of D2R in �-cells, we examined the expression ofD2R at the transcript level in human total pancreas andpurified cadaveric islets (Fig. 2). Using primers specific forboth long and short isoforms, short isoform alone, longisoform alone, and total D2R (Supplemental Table 1), weamplified cDNA prepared from pancreas, purified islets,and as a control, human striatum. We found that, similar

to striatum, D2RL was the predominantlyexpressed D2R isoform in human isletswith D2RS not visible under the experi-mental conditions. Furthermore, the sum ofthe transcripts derived from all the knownisoforms of D2R mRNA was higher in isletsrelative to total pancreas. The amount ofD2R message in human islets was compa-rable to that found in human striatum.

We used real-time PCR with the same setof primers to precisely quantitate levels ofspecific D2R isoforms (Fig. 3) in purifiedhuman islet, total human pancreas, and hu-man striatum cDNA. We found that 1) inhuman islets, the abundance of D2RL andD2RS transcripts is approximately 5-foldgreater than that found in human striatumand 50 times higher than that found in totalpancreas; 2) the amount of D2RL-specificmRNA paralleled the amount of total D2Rtranscripts; and 3) the copy number of D2RS-specific mRNA, although detectable, was lowand consistent with the semiquantitative resultsshown in Fig. 2 (data not shown).

In a separate set of experiments, weprobed cDNA, prepared from whole pan-creas, purified islets, and purified acinarexocrine tissue, for the expression of insulin(INS), as an islet purity control, and fourother products of the SLC (solute carrier)gene superfamily, SLC7A5 (also known asLAT1 or CD98 light chain), SLC3A2 (alsoknown as MDU1 or CD98), SLC18A2(also known as VMAT2), and SLC6A3(also known as DA reuptake transporter,DAT) relevant to a putative DA-mediatednegative feedback regulatory circuit of in-sulin secretion. As expected, both insulinand VMAT2 gene expression was more

than 30-fold enriched in islets relative to whole pancreasand more than 4 � 103-fold greater than the level mea-sured in purified exocrine tissue. We also found that isletswere enriched, relative to whole pancreas or pancreasexocrine tissue, for the expression of SLC7A5, SLC3A2,and SLC6A3.

Antagonism of monoamine transporters (MAT)and DA receptors enhances insulin secretion byhuman islets in vitro

The transport of monoamine neurotransmitters (e.g.DA) across cellular and vesicular membranes is mediatedby members of a family of transporters known as MAT.

FIG. 3. Expression of D2R, insulin (INS), SLC7A5, SLC3A2, SLC18A2, and SLC6A3mRNA in purified human cadaveric islets by quantitative RT-PCR. cDNA was amplifiedwith the primer pair 2hD2_F/2hD2R, amplifying all D2R-specific cDNA (D2R), the primerpair Long_hD2_F/Long_hD2_R specific for the long isoform of D2R (D2RL). The amountsof transcripts in each tissue were normalized to ACTB transcript content in therespective samples and the results for D2R are reported as relative to the amount ofspecific 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 ofspecific cDNA found in whole pancreas, which was arbitrarily set to 1. Error bars showthe SEM. The differences in expression of D2R between striatum, pancreas, and isletswere significant at the P � 0.05 level using Student’s t test. *, Significant differencefrom whole pancreas or exocrine tissue at the P � 0.05 level using Student’s t test.



In a previous report, we demonstrated that TBZ-medi-ated antagonism of VMAT2 resulted in enhanced GSIS ofisolated islets (25). To determine whether a similar effectmight 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 bemediated through DA depletion (26, 27), we also testedGSIS in the presence of DA. As an additional control, wetested the effects of haloperidol, a first-generation anti-psychotic and antagonist of DA action at D2R. We foundthat 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). Theamount of glucose-stimulated insulin release, normalizedto islet DNA content in the sample, in these static incubationassays was similar to that detected previously (28). As reportedfor rodent islets (29), we found that DA (1.0 �M) significantlyinhibited GSIS, whereas the antagonist of DA action at D2R,haloperidol, enhanced GSIS. Additional studies of GSIS usingislets from different donors confirmed the generality of ourfindings with DTBZ, DA, haloperidol, and the tyrosine hy-droxylase inhibitor AMPT (Fig. 5). As additional controls, wetested the effects of quinpirole (a selective D2 receptor agonist)and sulpiride (a selective D2 and D3 receptor antagonist) onhuman islet insulin secretion. As expected, agonism and antag-onism at D2R, respectively, decreased and increased GSIS inhuman islets (Fig. 5).

In the CNS behind the blood-brain barrier (BBB), DAreleased at the synapse is recycled into vesicles by theaction 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 reducestissue DA content (31). Because islets are not borderedby a BBB, yet are heavily vascularized and richly per-fused, we hypothesized that inhibition of DAT mightaffect a net loss of DA signaling and inhibition of DATby GBR 12909 might have similar effects on GSIS asdid DA depletion by DTBZ. We found that GBR 12909enhanced GSIS in human islets in vitro (Fig. 5), perhapsby blocking recycling and allowing DA to diffuse out ofthe cultured islet tissue. Supporting this idea was theadditional finding that islet tissue stimulated with 8 mM

glucose and treated with GBR 12909 contained less DAthan control islet tissue stimulated with 8 mM glucosealone (Fig. 6A).

On the basis of these results, we formed the workinghypothesis that DA stored in �-cell vesicles by action ofVMAT2 is coreleased into the extracellular space withinsulin upon glucose stimulation. DA acting at D2R in anautocrine manner or on nearby adjacent �-cells partiallysuppresses insulin secretion. Remaining DA moleculesnot 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 basalKRBB 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 (P � 0.0.05)from the wells with 8 mM glucose alone; **, significantly different from wells with 15 mM glucose alone; ***, significantly different from wellswith 3 mM glucose alone.



Antagonism of VMAT2 by TBZ enhances insulinsecretion in human islets in vitro by increasingsecretion pulse height

To better understand how this DA circuit might beoperating in islets, we examined the kinetics of GSIS un-der conditions of VMAT2 antagonism by DTBZ. Theinsulin concentration vs. time profile arising from perfu-sion of human islets with 1.5 mM glucose vs. 15 mM glu-cose with and without added DTBZ was measured andrevealed insulin pulses (Fig. 7). After the perfusate glucose

concentration was increased to 15 mM

from 1.5 mM, the insulin concentrationin collected fractions increased abovebasal levels. At 15 mM glucose, severalsignificant peaks of insulin secretion(averaging about 7 � 102 amol/ml perislet) were observed with an averageperiod of 5 � 0.5 min (SEM), consistentwith previous reports (32, 33). In thepresence of DTBZ, the amplitudes ofthe insulin pulses were significantly in-creased without a significant change intheir periodicity. The mass of insulinsecreted, as determined by the area un-der the curve during the high-glucoseperfusion, was higher in the presence ofDTBZ than in its absence, consistentwith the static incubation experiments(Fig. 5).

Human islets in vitro release DA ina glucose-dependent manner

In investigating a possible �-cell au-tocrine or paracrine circuit involvingDA-mediated regulation of GSIS andhaving shown that human islets and�-cells express D2R, and respond toDA and DA depletion in vitro, we nextexamined whether islets also releasedDA in response to glucose stimulation.Amperometry and voltammetry at mi-croelectrodes have been demonstratedto possess sufficient sensitivity andtemporal resolution to detect exocyto-sis of DA in the CNS (34). Similar tech-niques have been successfully appliedto measurements of 5-HT and acetyl-choline exocytosis from rodent and hu-man islets (6, 35–37), but release of DAhas 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 least50-fold more responsive to DA than to 5-HT.

Next, we probed the supernatant of islet cultures stim-ulated with glucose using an electrochemical techniqueand nCFM with preferential sensitivity to DA. The read-out of this chronoamperometric technique is a currentproportional to the DA concentration (Fig. 8C). Isletswere first incubated in KRBB without glucose, followedby KRBB supplemented with glucose to 1.5 mM and 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 against1.5 mM glucose, 15 mM glucose, and one or more of the indicated compounds. Each barrepresents the mean values obtained from three or more islet donors. For each donor andcondition tested, the GSIS (nanograms insulin per nanogram DNA) was normalized to meanGSIS obtained in 1.5 mM glucose for that donor. Error bars indicate the SEM. The mean GSISfor each compound tested was then compared with mean GSIS obtained in 15 mM glucosealone. *, Significantly different (P � 0.0.05) from the wells with 15 mM glucose alone;**, significantly different (P � 0.0.05) from the wells with 1.5 mM glucose alone.



to 15 mM glucose. The average currents detected underthese conditions were 21 � 2, 23 � 3, and 64 � 3 pA(mean � SEM), respectively. Although the observed av-erage current at 15 mM glucose was significantlygreater (P � 0.001) than the current at no glucose and1.5 mM glucose, the current detected at 1.5 mM glucosewas not significantly different from the current ob-served at no glucose, although there was evidence of anincreased number of current spikes, which may signifyevidence of pulsatile DA release. After incubation in 15mM glucose, the glucose concentration was reducednearly 30-fold to 0.5 mM, and we observed a corre-

sponding drop in the average current. Subsequently, weincreased the glucose concentration to 30 mM glucoseand observed a rise in the current. The average currentrose significantly above the baseline, but this time, wealso observed decay in the average current, possiblysignaling DA depletion in the cultured islets. To pro-vide evidence that the electrodes were still responsive toDA, at the end of the experiments, we added knownconcentrations of authentic exogenous DA to the isletcultures and measured the faradaic currents. On thisbasis, we estimate that DA release by islets was about102 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 theindicated concentration of DTBZ or GBR 12909, with islets placed within the well inserts. Islets were obtained from two or more different donorsas indicated by the value of n. At the end of the incubation, the supernatants were harvested and the DA and insulin content assayed. Islet tissuecontained within the inserts were washed twice and then harvested in 1 ml buffer and assayed for insulin, DA, and DNA. DA and insulinconcentration measurements were normalized to the DNA content of the well, and the mean value of at least triplicate wells was calculated. Tocompare groups, the amount of DA measured in the supernatant of each donor after stimulation with 8 mM glucose was set to represent 100%.All measurements in subsequent groups (supernatant vs. intracellular vs. treatment with GBR 12909) from that donor were expressed as thefraction of 8 mM glucose control supernatant value. B, DA release during perfusion experiments. Insulin and DA content were measured inselected 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 P � 0.05 level. C, DA uptake by culturedhuman islets. Islets were first cultured in 10 �M haloperidol to block [3H]DA binding to D2R. To these cultures 10 �M GBR 12909 or BCTP was thenadded to inhibit DAT function. [3H]DA was then added to the cultures for 1 h followed by washing and harvesting the islets. The amount of DAassociated 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 meanof triplicate wells. Error bars represent the SEM. The results are given as background-corrected normalized dpm or as a percentage of control usingthe mean dpm value from cultures with 1.5 mM glucose and no haloperidol or DAT inhibitors added as the denominator. *, Significantly different(P � 0.0.05) from the wells with 1.5 mM glucose alone; **, significantly different (P � 0.0.05) from the wells containing 1.5 mM glucose and 10�M haloperidol.



15 mM glucose 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 isletsin response to glucose stimulation, we measured the DAconcentration in supernatants obtained from the staticincubation experiments outlined above using the tech-nique of ELISA. Here DA-specific antibodies are used tocapture and detect DA from solution. The cross-reactivityof this assay with other endogenous monoamines (e.g.norepinephrine) was less than 1%. We found the amountof DA released by islets, normalized to the DNA contentof the well, was significantly greater in 15 mM glucoserelative to 1.5 mM glucose (mean concentration 0.28 �0.03 vs. 0.61 � 0.07 ng DA/ng DNA) (Fig. 6A). We nextmeasured both the DA and insulin content of the collected

fraction from islet perfusion experiments per-formed under stimulation by 8.0 mM glucose(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 uptakeby cultured human islets. Total islet uptake ofDA under the conditions tested was likely toreflect both binding and internalization byD2R and a component specifically mediatedby DAT. To discriminate between these com-ponents, total uptake was measured in Ringersbuffer with 1.5 mM glucose and the presenceand absence of a D2R antagonist (haloperidol)and one or both of two specific DAT inhibitors[GBR 12909 (38) or BTCP (39)] (Fig. 6C). Itwas observed that addition of GBR 12909and/or BCTP reduced the islet associated ra-dioactivity to about 60% of the control level.Haloperidol blocked up to 30% of the totaluptake of [3H] DA. When GBR 12909 orBCTP was added to the cultures containinghaloperidol, the internalized counts droppedan additional 35% to about 45% of the totalcontrol radioactivity and were significantly(P � 0.05) less than internalized counts ob-served in the presence of haloperidol blockingalone. Similar results were found in culturesperformed in 8.0 mM glucose.

Antagonism of VMAT2 or DAT reducesglucose excursions during IPGTT in theZucker obese T2D rat model in vivo

Our in vitro experiments suggested that themonoamine 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 12909in the Zucker fatty (ZF) rodent model of human pre-type2 diabetes (Fig. 9, top and middle panel). As an additionalcontrol, we examined the effects of GBR 12909 on bloodglucose levels during IPGTT in SD rats (Fig. 9, middlepanel). In vivo inhibition of both VMAT2 and DAT dur-ing IPGTT reduced the glucose excursions after ip glucosechallenge. The reduced glucose excursions measured afterGBR 12909 administration and 30 min after glucose chal-lenge were accompanied by increased serum insulin levelsin both the SD and ZF rodent models (Fig. 9, bottompanel). Previously, we reported similar effects on insulinsecretion 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 mM in the presence and absence ofDTBZ on the release of insulin from perfused human islets from two differentdonors. Upper panel shows results of 18 islets (donor a); bottom panel shows resultsof eight islets (donor b). Human islets were perfused (700 �l/min) in KRBB underbasal glucose conditions for 20 min followed by raising the perfusate glucoseconcentration to 15 mM glucose with and without added DTBZ. Insulinconcentrations were measured in duplicate or triplicate from 30-sec fractions of theperfusate. Cluster analysis (23) of the insulin concentration vs. time profile revealedthe significant peaks of insulin secretion (indicated by asterisks). **, Peaks obtainedduring perfusion in 15 mM glucose with DTBZ that were significantly larger ( P �0.05) than those obtained without DTBZ as determined by a sliding t test. The areaunder the curve was determined using the trapezoidal rule. Two representativeexperiments from a series of three are shown.



ATA enhance in vitro insulin secretion byhuman islets

The ATA (e.g. clozapine and olanzapine), are charac-terized by potent antagonist activity at DA D2 and D3and 5-HT2A receptors. In immunohistochemistry exper-iments, we demonstrated that D2R colocalized with insu-

lin on �-cells and that D2RL was thepredominant isoform expressed by�-cells. In static incubation experi-ments, we demonstrated that humanislet GSIS was inhibited by 5-HT, DA(1 �M), and the D2R selective agonistquinpirole and enhanced by D2R an-tagonists such as haloperidol andsulpiride. On this basis, we predictedthat ATA might also enhance humanislet GSIS. We performed static incuba-tion measurements of GSIS in the pres-ence of a range of concentrations ofeither clozapine or olanzapine using is-lets obtained from several donors (Figs.10 and 11). In general, we found thatboth clozapine and olanzapine signifi-cantly enhanced GSIS but that the ef-fect differed in magnitude from donorto donor (Fig. 11).

Discussion

The regulatory role of intrinsic inner-vation on islet insulin secretion hasbeen expertly summarized by Ahrén(3) and Thorens (40). Although paststudies have mostly relied on rodent is-lets, more recent microanatomicalstudies 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 acircuit may partially support overallregulation of GSIS given the observedrelative autonomy of human isletsfrom sympathetic innervation (5).

An appreciation of the presence ofmonoamine neurotransmitters withinislets and the role of these transmittersin 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) butalso that �-cell vesicles contained insulin and 5-HT (43,44) and/or DA as well. Next, it was found that �-cellsprobably express most if not all the enzymes needed for denovo synthesis of DA, its catabolism (13, 45, 46), and the

FIG. 8. Chronoamperometry of glucose-sensitive islet DA release. A, Input voltage waveformto the potentiostat (square wave pulses �0.08 to �0.220 V vs. Ag/AgCl with an 8-sec periodand 50% duty cycle). B, Output current signals from a nCFM in KRBB. The initial voltage pulsewas accompanied by a large increase in the double-layer current, which discharged wellbefore the end of 50% duty cycle mark. C, The faradaic current (i.e. generated from theoxidation of DA) was measured during the last second of the �220-mV pulse (gray bar in Aand B). The glucose concentration was varied as indicated, and the time vs. current data wereplotted (C) and the average currents at each glucose concentration calculated. At the end ofthe experiment (time � 2 h) authentic DA was added to the well to obtain the correspondingfaradaic current to confirm sensitivity to DA. Results are shown from a representativeexperiment in a series of three.



VMAT that sequester cytosolic neurotransmitters into se-cretory storage granules (11). In the human pancreas, theexpression of VMAT2 is largely confined to �-cell gran-ules containing insulin (11, 15). Low levels of VMAT2expression 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 toVMAT2, �-cells also express D2R, and as shown in thisreport, D2R expression in the pancreas is also largelyconfined to the �-cells. We found that in man, similar towhat was observed in the rat (13), islets predominantlyexpress the D2RL-specific isoform with D2RS-specifictranscript representing a minority. In the CNS, neuronalDA release can be down-regulated using DA receptor ago-nists via DA autoreceptors (8). Knockout experiments inmice reveal that this autoreceptor’s activity is most closelyassociated with the D2RS isoform (48). Additional studywill be needed to understand the molecular physiology ofislet DA receptor activity.

Based upon our earlier experiments, we hypothesizedthat DA, accumulated in �-cell vesicles by action ofVMAT2, is released into the extracellular space alongwith insulin upon glucose stimulation. DA acting at D2R,

FIG. 9. Top panel, TBZ reduces the blood glucose excursion duringIntraperitoneal Glucose Tolerance Test (IPGTT) in male ZF (obese)rats. Mean blood glucose values during IPGTT of ZF rats treatedwith vehicle alone (f, n � 4) or with TBZ at 2.25 �g/g body weight(�, n � 5). Error bars represent the SEM. The area under the curve(AUC) for each rodent’s blood glucose vs. time profile wascalculated using the trapezoid rule. The average AUC wascalculated and compared between control and TBZ-treated groups.The P value of the significance of the comparison of AUC by t test isshown. Middle panel, GBR 12909 reduces the blood glucoseexcursions during IPGTT in male ZF (obese) and SD rats. Mean bloodglucose values during IPGTT of rats treated with vehicle alone (Œand F, n � 5) or with GBR 12909 at 5.0 �g/g body weight (‚ andE, n � 5). The comparison of AUC was performed as detailedabove. Bottom panel, Sera collected at 30 min after IPGTT from theexperiments shown above were assayed for insulin. The meanserum insulin concentrations for treated rats were compared withthe values of untreated rats. The significance of comparison wasdetermined by t test and is indicated within the bar.

FIG. 10. Measurement of GSIS in islets from a single donor in thepresence of the ATA olanzapine. The islets were incubated in basalKRBB for 1 h and then seeded in wells and exposed to the indicatedglucose and drug concentration for 1 h. The mean insulinconcentration, normalized to the well DNA content, was thendetermined for sextuplicate wells. Mean insulin concentrations,normalized to the well DNA content, under different conditions werecompared with the mean response to 15 mM glucose alone by t test.The significance of each comparison, where P � 0.05, is shown. Errorbars indicate the SEM.



newly exposed at the cell surface after a round of GSIS orexpressed on vicinal �-cells, partially suppresses insulinsecretion and residual DA, not internalized with D2R orremoved by the circulation, would then be recycled by theaction 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 islettissue relative to whole pancreas (�6-fold) and at least10-fold greater than that measured in exocrine tissue. Wenext 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 contentwas reduced and GSIS was enhanced in the presence ofDTBZ. In addition, we found that inhibition of L-3,4-dihydroxyphenylalanine (L-DOPA, the direct precursorof DA) synthesis by AMPT, an inhibitor of tyrosine hy-droxylase, also enhanced islet GSIS. In this regard, it isinteresting to note that islets also express the large neutralamino acid transporter system, composed of a heavysubunit encoded the SLC3A2 gene and a light subunit encodedby the SLC7A5 gene. This heterodimer preferentially trans-ports branched-chain and aromatic (tryptophan and tyrosine)aminoacids, including L-DOPA(50), and ishighlyexpressed inbrain capillary endothelial cells that form the BBB (51). As a

consequence of the expression of DAT, the largeneutral amino acid transporter system andVMAT2, islet�-cellsappeartohavethecapacitytoremove DA and its biosynthetic precursors (e.g.L-DOPA) (52) from the circulation and concen-trate DA intravesicularly. As a direct consequenceof expression of these molecules by dopaminergicneurons, the concentration of DA at release siteshave been estimated to be about 1–100 �M, wellwithintheupper limitsofDAconcentrations testedhere. Nevertheless, it is not excluded that other in-travesicular monoamines (e.g. norepinephrine) areactivehere. InadditiontoblockingDAtransportatthe 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 12909enhanced islet GSIS. We speculated that inhibitionof 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 studyand attention to possible off-target effects isneeded.

We also found that human islet GSIS wasenhanced in the presence of antagonists ofD2R such as sulpiride and haloperidol. D2Ragonists such as exogenous DA and quin-pirole, as expected, diminished human islet

GSIS. A study of the kinetics of islet GSIS in the presenceof DTBZ revealed that enhanced insulin secretion wasprobably due to increases in the amplitude of pulsatilerelease rather than increases in the periodicity of insulinrelease. Lastly, we probed human islets in vitro for DAuptake mediated by DAT and glucose-dependent DA re-lease. We found that islets could internalize exogenous[3H]DA through a GBR 12909- or BTCP-sensitive path-way. We also observed that the amount of DA releasedinto 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 theadditional 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 circuitcould be manipulated to improve glucose clearance invivo using a pre-type 2 diabetes rodent model. We foundthat antagonism of VMAT2 reduced the in vivo glucoseexcursions after glucose tolerance testing and that inhibi-tion of DAT by GBR 12909 resulted in enhanced seruminsulin concentrations after an ip glucose challenge asmight be expected based on the results of our in vitro

FIG. 11. Measurement of GSIS in islets from multiple donors treated witholanzapine or clozapine. Static incubations were performed as indicated in Fig. 4.Each islet donor was tested against 15 mM glucose alone and 15 mM glucose plusthe indicated compounds and dose. For each donor and condition tested, the GSIS(nanograms insulin per nanogram DNA) was normalized to mean GSIS obtained in15 mM glucose alone for that donor to obtain the relative change. The mean relativechange of GSIS among all donors tested for each compound tested (black bar) wasthen compared with mean GSIS obtained in 15 mM glucose alone. The P valueobtained by Student’s t test, where P � 0.05, is shown for each condition tested inthe population.



experiments, although it is not excluded that these drugstarget sites beyond the endocrine pancreas. Althoughthese results suggest that off-label use of existing VMAT2inhibitors 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 secretioncould result in �-cell exhaustion (53). Consistent withthis, the too-much-of-a-good-thing hypothesis (54) wasproposed, in part, based on the results of the ADOPTstudy (55) showing that driving increased insulin secre-tion with sulfonylureas had worse long-term outcomesthan 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 withincreased risk of developing T2D. In the past, increasedrisk of developing T2D associated with ATA use has beenascribed to increases in adiposity (and consequent insulinresistance) mediated via their CNS effects on food intakeand physical activity (57, 58). We reasoned that an addi-tional contributor to overall risk might be that, if certainATA directly antagonize D2R on �-cells, an additionalincrease in insulin secretion might result through the DA/D2R-mediated autocrine feed-forward mechanism pro-posed above. We tested the effects of ATA clozapine andolanzapine on human islet GSIS in vitro and found thatGSIS in the presence of these ATA was indeed enhanced.Similar experiments measuring GSIS in rodent isletstreated with ATA have shown mixed results, rangingfrom 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 alsotested the effects of 5-HT on in vitro GSIS. As previouslyreported for rodent islets (62), we found that 5-HT inhib-ited islet GSIS, and thus it is reasonable to conclude that5-HT receptor antagonism would parallel the effects ofD2R antagonism. Thus, ATA via antagonism at D2R and5-HT receptors could potentially promote �-cell loss byallowing �-cells to produce more insulin (63, 64) withresultant endoplasmic reticulum stress (65). Lastly, theefficacy of bromocriptine, a potent D2R agonist, in themanagement of T2D (66) might also be explained via itslocal effects on �-cell insulin secretion in addition to itscentral 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 thepancreas. 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 bythree interlocking arterial circles (47). Reliance on suchsources of spillover DA would require that �-cells expressDAT. In this report, we have shown indirect evidence thatthat DAT is expressed in islets and has a role in regulatingGSIS in vitro and in vivo.

In this context, it is interesting to note that the brain isonly a minor source of peripheral DA. As is the case for5-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 postprandialspikes of serum DA in man (52) and that in vitro isletshave a functional DAT, suggests that DA in its role ofregulating �-cell insulin secretion represents an anti-in-cretin hypothesized to explain the beneficial effects ofbariatric surgery on T2D (69). Here surgical proceduresthat possibly lower circulating DA levels (e.g. sleeve gas-trectomy with duodenal switch) might be predicted tohave enhanced insulin secretion. Driving increased �-cellfunction, particularly when there is weight loss failure,could result in the eventual postoperative reoccurrence ofT2D as has been observed in a subpopulation of bariatricsurgery 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 168thStreet, BB 2006, New York, New York 10032. E-mail:[email protected].

This work was supported by the U.S. Public Health Service,National Institutes of Health, National Institute of Diabetes andDigestive 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.

References

1. Henquin JC 2000 Triggering and amplifying pathways of regula-tion of insulin secretion by glucose. Diabetes 49:1751–1760

2. Brunicardi FC, Shavelle DM, Andersen DK 1995 Neural regulationof the endocrine pancreas. Int J Pancreatol 18:177–195

3. Ahrén B 2000 Autonomic regulation of islet hormone secretion–implications for health and disease. Diabetologia 43:393–410

4. Cabrera O, Berman DM, Kenyon NS, Ricordi C, Berggren PO,Caicedo A 2006 The unique cytoarchitecture of human pancreaticislets has implications for islet cell function. Proc Natl Acad Sci USA103:2334–2339

5. Rodriguez-Diaz R, Abdulreda MH, Formoso AL, Gans I, Ricordi C,



Berggren PO, Caicedo A 2011 Innervation patterns of autonomicaxons in the human endocrine pancreas. Cell Metab 14:45–54

6. Rodriguez-Diaz R, Dando R, Jacques-Silva MC, Fachado A, Mo-lina J, Abdulreda MH, Ricordi C, Roper SD, Berggren PO, CaicedoA 2011 �-Cells secrete acetylcholine as a non-neuronal paracrinesignal priming �-cell function in humans. Nat Med 17:888–892

7. Wolf ME, Roth RH 1987 Dopamine neurons projecting to themedial prefrontal cortex possess release-modulating autoreceptors.Neuropharmacology 26:1053–1059

8. Westerink BH, de Vries JB 1989 On the mechanism of neurolepticinduced increase in striatal dopamine release: brain dialysis pro-vides direct evidence for mediation by autoreceptors localized onnerve terminals. Neurosci Lett 99:197–202

9. Maffei A, Liu Z, Witkowski P, Moschella F, Del Pozzo G, Liu E,Herold K, Winchester RJ, Hardy MA, Harris PE 2004 Identifica-tion of tissue-restricted transcripts in human islets. Endocrinology145:4513–4521

10. Kutlu B, Burdick D, Baxter D, Rasschaert J, Flamez D, Eizirik DL,Welsh N, Goodman N, Hood L 2009 Detailed transcriptome atlasof the pancreatic �-cell. BMC Med Genomics 2:3

11. Anlauf M, Eissele R, Schäfer MK, Eiden LE, Arnold R, Pauser U,Klöppel G, Weihe E 2003 Expression of the two isoforms of thevesicular monoamine transporter (VMAT1 and VMAT2) in theendocrine pancreas and pancreatic endocrine tumors. J HistochemCytochem 51:1027–1040

12. Henry JP, Botton D, Sagne C, Isambert MF, Desnos C, BlanchardV, Raisman-Vozari R, Krejci E, Massoulie J, Gasnier B 1994 Bio-chemistry and molecular biology of the vesicular monoamine trans-porter from chromaffin granules. J Exp Biol 196:251–262

13. Rubí B, Ljubicic S, Pournourmohammadi S, Carobbio S, ArmanetM, Bartley C, Maechler P 2005 Dopamine D2-like receptors areexpressed in pancreatic �-cells and mediate inhibition of insulinsecretion. J Biol Chem 280:36824–36832

14. Ericson LE, Håkanson R, Lundquist I 1977 Accumulation of do-pamine in mouse pancreatic B-cells following injection of L-DOPA.Localization to secretory granules and inhibition of insulin secre-tion. Diabetologia 13:117–124

15. Saisho Y, Harris PE, Butler AE, Galasso R, Gurlo T, Rizza RA,Butler PC 2008 Relationship between pancreatic vesicular mono-amine transporter 2 (VMAT2) and insulin expression in humanpancreas. J Mol Histol 39:543–551

16. Schmittgen TD, Livak KJ 2008 Analyzing real-time PCR data bythe comparative CT method. Nat Protoc 3:1101–1108

17. Weksler-Zangen S, Yagil C, Zangen DH, Ornoy A, Jacob HJ, YagilY 2001 The newly inbred cohen diabetic rat: a nonobese normo-lipidemic genetic model of diet-induced type 2 diabetes expressingsex differences. Diabetes 50:2521–2529

18. Pennington JM, Millar J, L Jones CP, Owesson CA, McLaughlinDP, Stamford JA 2004 Simultaneous real-time amperometric mea-surement of catecholamines and serotonin at carbon fibre ‘dident’microelectrodes. J Neurosci Methods 140:5–13

19. Gonon FG, Fombarlet CM, Buda MJ, Pujol JF 1981 Electrochem-ical treatment of pyrolytic carbon fiber electrodes. Anal Chem 53:1386–1389

20. Michael AC, Justice Jr JB 1987 Oxidation of dopamine and 4-meth-ylcatechol at carbon fiber disk electrodes. Anal Chem 59:405–410

21. Ahn SS, Blaha CD, Alkire MT, Wood E, Gray-Allan P, MarroccoRT, Moore WS 1991 Biphasic striatal dopamine release duringtransient ischemia and reperfusion in gerbils. Stroke 22:674–679

22. Crespi F, Cespuglio R, Jouvet M 1983 Differential pulse voltam-metry in brain tissue. III. Mapping of the rat serotoninergic raphenuclei by electrochemical detection of 5-HIAA. Brain Res 270:45–54

23. Veldhuis JD, Johnson ML 1986 Cluster analysis: a simple, versatile,and robust algorithm for endocrine pulse detection. Am J Physiol250:E486–E493

24. Tan S, Hermann B, Borrelli E 2003 Dopaminergic mouse mutants:

investigating the roles of the different dopamine receptor subtypesand the dopamine transporter. Int Rev Neurobiol 54:145–197

25. Raffo A, Hancock K, Polito T, Xie Y, Andan G, Witkowski P,Hardy M, Barba P, Ferrara C, Maffei A, Freeby M, Goland R,Leibel RL, Sweet IR, Harris PE 2008 Role of vesicular monoaminetransporter type 2 in rodent insulin secretion and glucose metabo-lism revealed by its specific antagonist tetrabenazine. J Endocrinol198:41–49

26. Leysen JE, Wynants J, Eens A, Janssen PA 1989 Ketanserin reducesa particular monoamine pool in peripheral tissues. Mol Pharmacol35:375–380

27. Reches A, Hassan MN, Jackson VR, Fahn S 1983 Lithium attenu-ates dopamine depleting effects of reserpine and tetrabenazine butnot that of � methyl-p-tyrosine. Life Sci 33:157–160

28. Kühtreiber WM, Ho LT, Kamireddy A, Yacoub JA, Scharp DW2010 Islet isolation from human pancreas with extended cold isch-emia time. Transplant Proc 42:2027–2031

29. Shankar E, Santhosh KT, Paulose CS 2006 Dopaminergic regula-tion of glucose-induced insulin secretion through dopamine D2receptors in the pancreatic islets in vitro. IUBMB Life 58:157–163

30. Kristensen AS, Andersen J, Jørgensen TN, Sørensen L, Eriksen J,Loland CJ, Strømgaard K, Gether U 2011 SLC6 neurotransmittertransporters: structure, function, and regulation. Pharmacol Rev63:585–640

31. Giros B, Jaber M, Jones SR, Wightman RM, Caron MG 1996Hyperlocomotion and indifference to cocaine and amphetamine inmice lacking the dopamine transporter. Nature 379:606–612

32. Hellman B, Salehi A, Gylfe E, Dansk H, Grapengiesser E 2009Glucose generates coincident insulin and somatostatin pulses andantisynchronous glucagon pulses from human pancreatic islets. En-docrinology 150:5334–5340

33. Song SH, Kjems L, Ritzel R, McIntyre SM, Johnson ML, VeldhuisJD, Butler PC 2002 Pulsatile insulin secretion by human pancreaticislets. J Clin Endocrinol Metab 87:213–221

34. Crespi F 1990 In vivo voltammetry with micro-biosensors for anal-ysis of neurotransmitter release and metabolism. J Neurosci Meth-ods 34:53–65

35. Aspinwall CA, Huang L, Lakey JR, Kennedy RT 1999 Comparisonof amperometric methods for detection of exocytosis from singlepancreatic �-cells of different species. Anal Chem 71:5551–5556

36. Smith PA, Duchen MR, Ashcroft FM 1995 A fluorimetric and am-perometric study of calcium and secretion in isolated mouse pan-creatic �-cells. Pflugers Arch 430:808–818

37. Barbosa RM, Silva AM, Tome AR, Stamford JA, Santos RM, Ro-sario LM 1998 Control of pulsatile 5-HT/insulin secretion fromsingle mouse pancreatic islets by intracellular calcium dynamics.J Physiol 510(Pt 1):135–143

38. Matecka D, Rothman RB, Radesca L, de Costa BR, Dersch CM,Partilla JS, Pert A, Glowa JR, Wojnicki FH, Rice KC 1996 De-velopment of novel, potent, and selective dopamine reuptakeinhibitors through alteration of the piperazine ring of 1-[2-(di-phenylmethoxy)ethyl]-and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR12909). J Med Chem 39:4704 – 4716

39. He X, Raymon LP, Mattson MV, Eldefrawi ME, de Costa BR 1993Further studies of the structure-activity relationships of 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine. Synthesis and evaluation of1-(2-benzo[b]thienyl)-N,N-dialkylcyclohexylamines at dopamineuptake and phencyclidine binding sites. J Med Chem 36:4075–4081

40. Thorens B 2010 Central control of glucose homeostasis: the brain-endocrine pancreas axis. Diabetes Metab 36(Suppl 3):S45–S49

41. Falck B, Hellman B 1964 A fluorescent reaction for monoamines inthe insulin producing cells of the guinea-pig. Acta Endocrinol (Co-penh) 45:133–138

42. Cegrell L, Falck B, Rosengren AM 1967 Dopamine and 5-hydroxy-tryptamine in the guinea-pig pancreas. Life Sci 6:2483–2489



43. Jaim-Etcheverry G, Zieher LM 1968 Electron microscopic cyto-chemistry of 5-hydroxytryptamine (5-HT) in the �-cells of guineapig endocrine pancreas. Endocrinology 83:917–923

44. Zawalich WS, Tesz GJ, Zawalich KC 2001 Are 5-hydroxytrypta-mine-preloaded �-cells an appropriate physiologic model systemfor establishing that insulin stimulates insulin secretion? J BiolChem 276:37120–37123

45. Ito K, Hirose H, Maruyama H, Fukamachi S, Tashiro Y, Saruta T1995 Neurotransmitters partially restore glucose sensitivity of in-sulin and glucagon secretion from perfused streptozotocin-induceddiabetic rat pancreas. Diabetologia 38:1276–1284

46. Iturriza FC, Thibault J 1993 Immunohistochemical investigation oftyrosine-hydroxylase in the islets of Langerhans of adult mice, ratsand guinea pigs. Neuroendocrinology 57:476–480

47. Mezey E, Eisenhofer G, Harta G, Hansson S, Gould L, Hunyady B,Hoffman BJ 1996 A novel nonneuronal catecholaminergic system:exocrine pancreas synthesizes and releases dopamine. Proc NatlAcad Sci USA 93:10377–10382

48. Usiello A, Baik JH, Rougé-Pont F, Picetti R, Dierich A, LeMeur M,Piazza PV, Borrelli E 2000 Distinct functions of the two isoforms ofdopamine D2 receptors. Nature 408:199–203

49. Bannon MJ 2005 The dopamine transporter: role in neurotoxicityand human disease. Toxicol Appl Pharmacol 204:355–360

50. Kageyama T, Nakamura M, Matsuo A, Yamasaki Y, Takakura Y,Hashida M, Kanai Y, Naito M, Tsuruo T, Minato N, ShimohamaS 2000 The 4F2hc/LAT1 complex transports L-DOPA across theblood-brain barrier. Brain Res 879:115–121

51. Boado RJ, Li JY, Nagaya M, Zhang C, Pardridge WM 1999 Selec-tive expression of the large neutral amino acid transporter at theblood-brain barrier. Proc Natl Acad Sci USA 96:12079–12084

52. Goldstein DS, Swoboda KJ, Miles JM, Coppack SW, Aneman A,Holmes C, Lamensdorf I, Eisenhofer G 1999 Sources and physio-logical significance of plasma dopamine sulfate. J Clin EndocrinolMetab 84:2523–2531

53. Hansen JB, Arkhammar PO, Bodvarsdottir TB, Wahl P 2004 Inhi-bition of insulin secretion as a new drug target in the treatment ofmetabolic disorders. Current medicinal chemistry 11:1595–1615

54. Aston-Mourney K, Proietto J, Morahan G, Andrikopoulos S 2008Too much of a good thing: why it is bad to stimulate the �-cell tosecrete insulin. Diabetologia 51:540–545

55. Kahn SE, Haffner SM, Heise MA, Herman WH, Holman RR, JonesNP, Kravitz BG, Lachin JM, O’Neill MC, Zinman B, Viberti G2006 Glycemic durability of rosiglitazone, metformin, or glyburidemonotherapy. N Engl J Med 355:2427–2443

56. Nord M, Farde L 2011 Antipsychotic occupancy of dopamine re-ceptors in schizophrenia. CNS Neurosci Ther 17:97–103

57. Elias AN, Hofflich H 2008 Abnormalities in glucose metabolism inpatients with schizophrenia treated with atypical antipsychoticmedications. Am J Med 121:98–104

58. Holt RI, Peveler RC 2006 Antipsychotic drugs and diabetes: anapplication of the Austin Bradford Hill criteria. Diabetologia 49:1467–1476

59. Johnson DE, Yamazaki H, Ward KM, Schmidt AW, Lebel WS,Treadway JL, Gibbs EM, Zawalich WS, Rollema H 2005 Inhibi-tory effects of antipsychotics on carbachol-enhanced insulin secre-tion from perifused rat islets: role of muscarinic antagonism inantipsychotic-induced diabetes and hyperglycemia. Diabetes 54:1552–1558

60. Melkersson K 2004 Clozapine and olanzapine, but not conven-tional antipsychotics, increase insulin release in vitro. Eur Neuro-psychopharmacol 14:115–119

61. Melkersson K, Khan A, Hilding A, Hulting AL 2001 Differenteffects of antipsychotic drugs on insulin release in vitro. Eur Neu-ropsychopharmacol 11:327–332

62. Lindström P, Sehlin J 1983 Opposite effects of 5-hydroxytrypto-phan and 5-hydroxytryptamine on the function of microdissectedob/ob-mouse pancreatic islets. Diabetologia 24:52–57

63. Grill V, Björklund A 2009 Impact of metabolic abnormalities for�-cell function: clinical significance and underlying mechanisms.Mol Cell Endocrinol 297:86–92

64. Chang-Chen KJ, Mullur R, Bernal-Mizrachi E 2008 �-Cell failureas a complication of diabetes. Rev Endocr Metab Disord 9:329–343

65. Qian L, Zhang S, Xu L, Peng Y 2008 Endoplasmic reticulum stressin �-cells: latent mechanism of secondary sulfonylurea failure intype 2 diabetes? Med Hypotheses 71:889–891

66. Meier AH, Cincotta AH, Lovell WC 1992 Timed bromocriptineadministration reduces body fat stores in obese subjects and hyper-glycemia in type II diabetics. Experientia 48:248–253

67. Kirchgessner AL, Gershon MD 1990 Innervation of the pancreas byneurons in the gut. J Neurosci 10:1626–1642

68. Eisenhofer G, Aneman A, Friberg P, Hooper D, Fåndriks L, Lon-roth H, Hunyady B, Mezey E 1997 Substantial production of do-pamine in the human gastrointestinal tract. J Clin EndocrinolMetab 82:3864–3871

69. Rubino F, R’bibo SL, del Genio F, Mazumdar M, McGraw TE2010 Metabolic surgery: the role of the gastrointestinal tract indiabetes mellitus. Nature reviews Endocrinology 6:102–109

70. DiGiorgi M, Rosen DJ, Choi JJ, Milone L, Schrope B, Olivero-Rivera L, Restuccia N, Yuen S, Fisk M, Inabnet WB, Bessler M2010 Re-emergence of diabetes after gastric bypass in patients withmid- to long-term follow-up. Surg Obes Relat Dis 6:249–253



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