Abstract
The present studies extend recent findings that mice null for the 2A adrenergic receptor ( 2A AR KO mice) lack suppression of exogenous secretagogue-stimulated insulin secretion in response to 2 AR agonists by evaluating the endogenous secretagogue, glucose, ex vivo, and providing in vivo data that baseline insulin levels are elevated and baseline glucose levels are decreased in 2A AR KO mice. These latter findings reveal that the 2A AR subtype regulates glucose-stimulated insulin release in response to endogenous catecholamines in vivo. The changes in 2A AR responsiveness and resultant changes in insulin/glucose homeostasis encouraged us to utilize proteomics strategies to identify possible 2A AR downstream signaling molecules or other resultant changes due to perturbation of 2A AR expression. Although agonist stimulation of islets from wild type (WT) mice did not significantly alter islet protein profiles, several proteins were enriched in islets from 2A AR KO mice when compared with those from WT mice, including an enzyme participating in insulin protein processing. The present studies document the important role of the 2A AR subtype in tonic suppression of insulin release in response to endogenous catecholamines as well as exogenous 2 agonists and provide insights into pleiotropic changes that result from loss of 2A AR expression and tonic suppression of insulin release.
Introduction
Three subtypes of 2 adrenergic receptors (AR; 2A , 2B and 2C ) (Ruffolo & Hieble 1994 , Docherty 1998 ) are known to regulate multiple signaling pathways by coupling to G i and G o proteins. Although pharmacological studies are consistent with the interpretation that the 2A AR subtype attenuates glucose-stimulated insulin release from pancreatic cells (Ullrich & Wollheim 1985 , 1989a , Niddam et al. 1990 , and both 2A AR and 2C AR are expressed in a FACS-purified rat -cell population (Chan et al. 1997) , the lack of 2 AR subtype-specific ligands has prevented definitive identification of the subtype involved in suppression of insulin release. The development of mice null for each of these subtypes (Link et al. 1995 , 1996 , Altman et al. 1999 ) has helped to elucidate the subtype-specific role of each 2 AR subtype in vivo. Recent studies in fact implicate the 2A AR in mediating suppression of insulin release in response to forskolin and IBMX, agents which elevate cAMP content in islets (Peterhoff et al. 2003) .
Introduction
The molecular basis for 2A AR-mediated suppression of insulin release remains unknown. Recent studies suggest that the 2A AR, but not the 2C AR, is coupled to inhibition of adenylyl cyclase and hyperpolarization via K + currents in mouse islets (Peterhoff et al. 2003) . Nonetheless, the known effectors of 2 AR actions, i.e. decrease in adenylyl cyclase activity (Katada & Ui 1981 , Yamazaki et al. 1982 , increase in receptor-operated K + currents, decrease in voltage-gated Ca 2+ currents (Limbird 1988) and modulation of MAP kinase activity (Richman & Regan 1998) do not appear to explain the ability of 2 AR to suppress glucose-stimulated insulin release. For example, regulation of cAMP content cannot explain 2A AR-mediated inhibition of insulin release from pancreatic cells, since 2 AR agonists inhibit insulin release even when dibutyryl cAMP is the secretagogue (Rabinovitch et al. 1978 , Ullrich & Wollheim 1984 , indicating that suppression of cAMP production cannot be the principal mechanism for 2A AR modulation of insulin release (Debuyser et al. 1991 , Sharp 1996 . Similarly, although insulin secretion can be inhibited by G-protein mediated direct regulation of K + and Ca 2+ channels (Nilsson et al. 1989 , Drews et al. 1990 , Debuyser et al. 1991 , it is also observed that 2 AR agonists, along with somatostatin and galanin, can inhibit both ionomycin-induced insulin secretion and secretion in permeabilized cells where transmembrane potential and ionic gradients are disrupted (Jones et al. 1987 , Ullrich & Wollheim 1988 , 1989b , Ullrich et al. 1990 , Lang et al. 1995 . So it is unlikely 2 AR activation of K + currents or suppression of Ca 2+ currents can fully account for 2 AR attenuation of insulin release. The 2 AR also has been demonstrated to stimulate MAP kinase activity in freshly isolated vascular smooth muscle cells (Richman & Regan 1998 ), but there is no direct evidence to date of a role for regulation on MAP kinase in inhibition on insulin release. However, it has been show that stimulation of MAP kinase is not essential for activation of insulin release (Khoo & Cobb 1997) .
Introduction
The present study was undertaken for two purposes. First, we wished to examine the impact of the natural secretagogue, glucose, on stimulating insulin release from wild type (WT) vs 2A AR 'knockout' (KO) mice, and the regulation of glucose-stimulated insulin release (GSIS) by 2 agonist. Secondly, we explored whether a proteomic strategy would successfully identify downstream signaling targets of the 2A AR as well as the pleotropic consequences of having no 2A AR expression. We chose a proteomic strategy because comparing the protein profiles of islets from WT and KO mice using high-capacity two-dimensional gel electrophoresis resolves thousands of proteins of wide molecular weight range and permits identification of changes in posttranslational states of proteins (Wilm et al. 1996) , ultimately providing an unbiased examination of changes in protein expression or modulation as a consequence of changes in 2A AR function.
Materials and methods

Experimental animals
All animal procedures were approved in advance by our institutional IACUC committee; the WT mice used were female C57BL6J strain provided by Charles River Laboratories, Inc. (Wilmington, MA, USA). The 2A AR KO mice were backcrossed onto this strain to achieve 98·44% genomic identity. Mice were 11-12 weeks old. Animals were housed in AALAAC-approved facilities. The authors affirm that these studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health.
Materials
RPMI Medium 1640, Hank's balanced salt solution (HBSS) and 10% fetal bovine serum (FBS) was obtained from Invitrogen (Carlsbad, CA, USA). Penicillin and streptomycin are from Gibco (Carlsbad, CA, USA). Collagenase P was from Roche (Basel, Switzerland). 0·5% IPG (Immobilized pH gradient) buffer pH4-7 was purchased from (Amersham Biosciences, Piscataway, NJ, USA). All other materials were reagent grade from Sigma. 2A AR KO mice were of the same age and genetic background, provided by Brian Kobilka (Stanford University, Stanford, CA, USA). For insulin content assay, the mice used were males of C57BL6J strain of 1 year old, kindly provided by Brian Kobilka. Animals were housed in AALAAC-approved facilities.
Islet isolation and culture
The protocol for islet isolation (Brissova et al. 2002) followed previously described procedures (Stefan et al. 1987 , Rocheleau et al. 2002 . The entire abdominal cavity of the mouse was exposed by a bilateral subcostal incision that was extended into a T incision by a midline transverse incision. The pancreas was dissected from its attachment to the spleen and intestine, the blood supply severed, and the pancreas removed and immediately placed into cold HBSS before killing the mouse by rapid cervical dislocation.
Islet isolation and culture
The pancreas was washed with ice-cold HBSS, diced into 1 mm pieces, and suspended in approximately 50 ml of ice-cold HBSS. After sitting for 1 min, any excess fatty tissue was removed. Each pancreas was placed into a 7 ml siliconized glass test tube containing 5 ml cold HBSS, to which 12 mg of collagenase P was added. The tube was then sealed and submerged in a 37 C water bath and rapidly shaken for 12 min. The tube was centrifuged in a benchtop Dynac centrifuge (BD Bioscience, Franklin Lakes, NJ, USA) at the highest speed for 30 s. The supernatant was discarded and the pellet resuspended in HBSS at 4 C. Washing was continued until the supernatant was clear; the islets were then poured into an untreated 100 mm Petri-dish with 25 m1 of islet medium (composition: RPMI 1640, 11 mM glucose, 10% FBS, penicillin/streptomycin 100 U/ml). The islets were purified by individually transferring islets into a dish of clean islet medium using a micropipette under a dissecting microscope. Islets were cultured in untreated culture dishes overnight in islet medium maintained at 37 C and 5% CO 2 .
Measurement of insulin secretion by islet perifusion
A cell perifusion system was used to analyze islet insulin secretion at different concentrations of glucose (Wang et al. 1997 , Rocheleau et al. 2002 . Totally 40 mouse pancreatic islets selected after overnight culture, as described above, were placed in each column. Islets were equilibrated in DMEM containing 2·8 mM glucose, and 10 mM HEPES (pH 7·4) and 0·2% bovine serum albumin (BSA) (Sigma) at 37 C. To control for differences in islet size, islets of comparable size were selected from WT and KO mice, using the ruler in the dissecting microscope screen. Islet function was analyzed by raising the extracellular glucose concentration from 2·8 to 16·8 mM and monitoring insulin secretion in response to the glucose concentration change. A flow rate of 1 ml/min was used with each fraction containing 3 ml of effluent. Collection of samples for insulin assay began 20 min after the initial equilibration in 2·8 mM glucose. Insulin content of each fraction was assessed using radioimmunoassays (RIA), performed in duplicate on each fraction using insulin antibody-coated tubes (INC Pharmaceuticals Inc., Costa Mesa, CA, USA) (Wang et al. 1997) .
Islet extraction and 2D difference gel electrophoresis (2D-DIGE)
Islets were hand-picked to purity from pancreatic exocrine tissue, cultured overnight, counted and transferred by a micro pipette into a 1·5 ml Eppendorf tube (Eppendorf, Westbury, NY, USA). For the experiment to compare islet samples from WT and 2A AR KO mice, the supernatant was removed after a brief spin at 16 000 g, 4 C for 5 s, and islets were immediately deep frozen in liquid nitrogen. Islets of comparable size were selected as above. In experiments comparing control and agonist-treated islets from WT mice, each collection was pooled in 0·45 ml of islet medium (as above), and incubated for 5 min at 37 C with 10 6 M dexmedetomidine or not (control group) prior to freezing the samples in liquid nitrogen. After this 5 min treatment, islets were snap frozen as described above. Storage of islet extracts was at 80 C.
Islet extraction and 2D difference gel electrophoresis (2D-DIGE)
Mouse pancreatic islets (typically 300-450 per experimental condition obtained by harvesting, 30-50 islets per mouse) were pooled in sample lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, 5 mM magnesium acetate) prior to labeling with 200 pmol of either Cy3 or Cy5 (Amersham Biosciences, Piscataway, NJ, USA) for 30 min on ice in the dark. These reactions were quenched with 2 µl of 10 mM lysine for 10 min on ice in the dark. The quenched Cy3-and Cy5-labeled samples were combined and added to an equal volume of 2x rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 4 mg/ml DTT) supplemented with 0·5% IPG (Immobilized pH gradient) buffer 4-7. The stoichiometry of Cy-dye labeling was purposefully low to ensure equal labeling of the same proteins from each extract; the dyes exhibit a sensitivity that is at least that of conventional silver stain (1 ng) under these conditions (Tonge et al. 2001) . Preliminary experiments were consistent with previous reports that differences in proteins identified from WT vs 2A AR KO mice or /+ agonist treated islets from WT mice were indistinguishable whether the samples were labeled with either Cy3 or Cy5 (Tonge et al. 2001 , Gharbi et al. 2002 .
Islet extraction and 2D difference gel electrophoresis (2D-DIGE)
The labeled protein extracts were separated by standard 2D gel electrophoresis using an IPGphor first-dimension isoelectric focusing unit (Amersham Biosciences) and 24 cm 4-7 immobilized pH gradient strips (Amersham Biosciences), followed by seconddimension 12% SDS-PAGE using an Ettan DALT 12 unit (Amersham Biosciences). Each 2D gel typically separated protein extract from 600 islets total. The estimated protein content applied to each gel is 400 µg. The isoelectrically focused samples were reduced and alkylated with DTT and iodoacetamide and equilibrated into the second dimension loading buffer (6 M Urea, 30% glycerol, 2% SDS, 50 mM Tris pH 8·8) per the manufacturer's protocol. Second-dimension SDS PAGE gels were precast with low-fluorescence glass plates, with one glass plate pre-silanized (Bind-silane; Amersham Biosciences) to affix the polymerized gel to only one of the glass plates.
Islet extraction and 2D difference gel electrophoresis (2D-DIGE)
After 2D gel electrophoresis, the entire gel was imaged to detect the Cy3-and Cy5-conjugated protein spots on each gel using mutually exclusive excitation/ emission wavelengths of 520/590 nm for Cy3 and 620/680 nm for Cy5 using a 2D 2920 Master Imager (Amersham Biosciences). Individual protein spotfeatures were co-detected and analyzed using DeCyder Differential In-gel Analysis software (Amersham Biosciences), where individual spot volume ratios were calculated for each protein-pairs. Two standard deviations of the mean, calculated from a modeled normal distribution of all spot volume ratios, was used to identify those protein spot features that exhibited significant abundance changes within the 95 th percent confidence level.
Islet extraction and 2D difference gel electrophoresis (2D-DIGE)
The 2D gels were subsequently stained with Sypro Ruby (Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions, to allow for accurate robotic protein excision. Since the CyDye labeling is purposefully limited to allow for quantification of abundance changes (see above), this post-stain visualizes 97% of the unlabeled protein which may exhibit a different migration during SDS-PAGE due to the increased molecular weight and hydrophobicity of the Cy-dyes. Sypro Ruby images were acquired on the same imager using 400/633 nm wavelengths, as well as re-imaged post-excision to ensure accurate protein excision.
Islet extraction and 2D difference gel electrophoresis (2D-DIGE)
For the data described in Table 1 , the approximate fold change shown in protein content is calculated by pooling islet extracts from islets from 10 mice of each (WT, KO) genotype to normalize for mouse to mouse and islet to islet variability. We know from preliminary experiments that the same sample run in duplicate varies less than 5% in signal, and that the signal changes are not different if the Cy-3 and Cy-5 are alternated between WT vs KO samples. The particular study described in Table 1 was repeated three times, and the profile was similar for all three experiments, based on visual examination. The quantitation, however, occurred for one of three experiments performed in an identical fashion, as samples in the two replicate experiments were not conjugated with Cy-dyes, so that samples excised from the gel could be subsequently analyzed by mass spectrometry to obtain masses for each of the proteolytic fragments corresponding to spots of interest (see below).
Protein identification by mass spectrometry and database searching
Protein spots of interest were robotically excised, equilibrated with 100 mM ammonium bicarbonate and dehydrated with acetonitrile in a 96-well plate format using Ettan Spot Picker and Digester workstations (Amersham Biosciences). Dehydrated gel plugs were digested in-gel with 10 µl porcine modified trypsin protease (Promega, Madison, WI, USA) in 25 mM ammonium bicarbonate for 2 h at 37 C, and tryptic peptides were extracted in two cycles of 60% a Promotes the formation of large chylomicron through a mechanism of ceramide hydrolysis (Kirby et al. 2002) ; b rate limiting enzyme of serine synthesis from 3-phospoglycerate (Yamasaki et al. 2001) ; c catalyzing the reaction from long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen (Lowe et al. 1989); carboxypeptidase B as well as trypsin is involved in the coversion from proinsulin to insulin (Kemmler et al. 1971 ); a serum marker used for acute pancreatitis and dysfunctions of pancreatic transplants (Yamamoto et al. 1992) ; e serpins (serine protease inhibitors) are human homologs of this protein and studies on serpins revealed they inhibit proteases by a mechanism of suicide substrate inhibition (Benarafa et al. 2002) ; f functional annotation indicates it has multiple peptidase activities, including trypsin and chymotrypsin activity, as well as serine-type endopeptidase activity (Okazaki et al. 2002) ; fold change estimated from a sample created by pooling islets isolated from ten mice of each genotype, with the intention of normalizing for mouse to mouse and islet to islet variability. Search algorithms are described in Materials and methods. *, inferred from electronic annotation (IEA); §, inferred from sequences or structural similarity (ISS); ‡, inferred from mutant phenotype (IMP). ms-ms indicates tandem mass spectrometry.
Protein identification by mass spectrometry and database searching
acetonitrile, 0·1% trifluoroacetic acid and dried by vacuum centrifugation. Peptides were reconstituted in 10 µl of 0·1% triflouroacetic acid, and manually de-salted/concentrated into 2 µl of 60% acetonitrile, 0·1% triflouroacetic acid using C18 ziptip pipette tips (Millipore, Billerica, MA, USA).
Protein identification by mass spectrometry and database searching
The peptide eluate (0·5 µl) was applied to a MALDI target and overlaid with 0·5 µl of cyano 4-hydroxycinnamic acid matrix (10 mg/ml in 60% acetonitrile, 0·1% triflouroacetic acid). Matrix-assisted laser desorption/ionization, time-of-flight (MALDI-TOF) mass spectrometry was performed on a Voyager 4700 (Applied Biosystems, Foster City, CA, USA). Peptide mass maps were acquired in reflectron mode (20 keV accelerating voltage) with 125 nsec delayed extraction, averaging 2000 laser shots per spectrum, and internally calibrated to within 20 ppm mass accuracy using trypsin autolytic peptides (m/z=842·51, 1045·56 and 2211·10). Ions specific for each sample (discrete from background and trypsin-derived ions) were used to interrogate mouse sequences entered in the SWISS-PROT and NCBInr databases using the MASCOT (www.matrixscience.com) and ProFound (prowl.rockefeller.edu) database search algorithms, respectively. Protein identifications from MALDI-TOF peptide mass maps are based on the masses of the tryptic peptides (Henzel et al. 1993 , Yates et al. 1993 . Searches were performed without constraining protein molecular weight or isoelectric point, and allowed for carbamidomethylation of cysteine, partial oxidation of methionine residues, and one missed trypsin cleavage.
Homology analysis and functional annotation
Human genomic BLAST searches from the NCBI website (http://www.ncbi.nlm.nih.gov/genome/seq/ page.cgi?F=HsBlast.html&&ORG=Hs) were used to compare the homology between assigned murine proteins and their human homologs, using the protein database and blastp program. The Genbank accession numbers for murine and human homologs allowed us to explore known functions or activities of these proteins. The functional annotation of each protein was provided by the Gene Ontology Consortium. Evidence supporting the annotation can be categorized into IC (inferred by curator), IDA (inferred from direct assay), IEA (inferred from electronic annotation), IEP (inferred from expression pattern), IGI (inferred from genetic interaction), IMP (inferred from mutant phenotype), IPI (inferred from physical interaction), ISS (inferred from sequence or structural similarity), NAS (non-traceable author statement), and/or TAS (traceable author statement) (http://www.geneontology.org/doc/GO.evidence.html). The evidence utilized to assign function to the protein spots identified is given in Table 1 .
BIOCARTA pathway analysis
BIOCARTA pathway analysis was performed to learn whether proteins identified as differentially expressed in WT vs 2A AR KO islets were associated with linked metabolic or signaling pathways. The BIOCARTA pathways are represented by a 0/1 matrix. Locus Link IDs of these proteins identified were filled into the rows of the matrix, in comparison with the available BIOCARTA pathways listed in columns. Two matrices were generated for mouse and human Locus Link IDs each. When a protein identified is involved in an available BIOCARTA pathway, the intersected slot of related protein and pathway was valued 1, otherwise 0. The analysis was performed on the mouse genes as well as on the human homologs. We identified several pathways in which the proteins identified in our proteomic study were involved.
Analysis of genomic DNA

PCR amplification of relevant DNA
Two primers, 5 -CTAGCTGGCTGACTGTTCCTT TGT-3 and 5 -TCCTGCTTTGGCTTTCTGAG-3 were selected to PCR amplify a 401 bp fragment of the coding region (exon 8) of the mouse Bpnt1 gene locus (Locus Link ID: 23827). Genomic DNA was obtained from C57BL6 WT and 2A AR KO mice using the rodent tail protocol using the DNeasy Tissue Kit (Qiagen Inc., Valencia, CA, USA). Genomic DNA from R1 embryonic stem (ES) cells was extracted from 2 10 7 cells. Next 165 ng of genomic DNA from each sample was added to a reaction tube containing 0·5 µg of each primer, 1 xPfu reaction buffer (Stratagene, La Jolla, CA, USA), 0·25 mM of each dNTP, 2 µl DMSO (Sigma), and 2·5 units of Pfu Turbo DNA polymerase (Stratagene) in a total reaction volume of 40 µl. PCR amplification was achieved using a PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA, USA) with a 94 C initial denaturation for 2 min, followed by 29 cycles of 94 C for 45 s, 57·3 C for 45 s, and 72 C for 1 min 45 s. The PCR reaction was followed by prolonged incubation (6 h) at 4 C. Samples were analyzed by running 10 µl of the product on a 1·2% agarose gel containing ethidium bromide and photographed under UV light by a Geldoc 2000 unit (Biorad, Hercules, CA, USA), analyzed by Quantity One software (Biorad).
PCR amplification of relevant DNA
The 401 bp PCR product was resolved in a 1·2% agarose gel at 100 V for 45 min; ion exchange cellulose paper (DE81, from Whatman, Kent, UK) was inserted below the desired PCR fragment on the gel, and the gel was submitted to an additional 30 min of electrophoresis. Paper with the transferred DNA fragment was washed four times with 100 µl 'tombstone buffer' (20% ethanol, 1 M LiCl, 10 mM Tris pH 7·6, 1 mM EDTA).
PCR amplification of relevant DNA
The DNA sample was extracted and precipitated by transfer of the paper sample to a solution containing 2 µl of glycogen (30 mg/ml) in 1 ml 100% ethanol for 15 min on dry ice. The DNA was isolated by centrifugation at 16 000 g on a Sorvall biofuge pico centrifuge (Kendro laboratory products, Newtown, CT, USA). The pellet was washed with 70% ethanol and resuspended into 10 µl of 10 mM Tris-HCl, pH 8·0, 1 mM EDTA.
Restriction enzyme analysis of the purified PCR product
The purified DNA fragment (10 µl containing approximately 0·4 µg of DNA) was digested with Hha I (20 U/incubation) (New England Biolabs, Beverly, MA, USA) at 37 C overnight and the digested fragments were separated on a 4% agarose gel.
DNA sequence analysis
After purification, 10 µl of 25 ng/µl amplified 401 bp PCR product from each source described above were sequenced using the ABI PRISM dye terminator sequencing reaction (Perkin-Elmer). Ten nano grams/ micro liter of primer 5 -CTAGCTGGCTGACTGTT CCTTTGT-3 was used for sequencing using an automated sequencer (ABI PRISM 377, Perkin-Elmer) and the analysis was performed using sequence analysis software. Sequencing was performed at the Vanderbilt DNA sequencing shared resource (www.mc.vanderbilt. edu/vicc/showcontent.php?id=319).
Quantitation of plasma insulin and glucose levels and pancreatic insulin content in WT vs 2A AR KO mice

Blood glucose measurement
Blood was collected from the saphenous vein of 10-week old female mice littermates using a 25 gauge needle connected to a Lithium-Heparin CB 300 Microvette (Sarstedt, Numbrecht, Germany). Mice were fasted for 4 h before evaluation. For each glucose assay 10 µl of blood was used. Glucose analysis was performed on a Hemocue B-Glucose analyzer with microcuvettes (HemoCue Inc., Lake Forest, CA, USA).
Plasma insulin measurement
Blood, collected from the same mice fasted for 4 h, was centrifuged at 16 000 g in a Heraeus Biofuge fresco benchtop centrifuge (Kendro, Asheville, NC, USA). Plasma was collected and assayed by radioimmunoassay for insulin according to published protocols (Brissova et al. 2002) . Pancreatic islet insulin content was determined as previously described (Brissova et al. 2002) .
Results

Inhibition of glucose-stimulated insulin release by the 2 AR agonist UK 14 304 is eliminated in 2A AR KO islets
Glucose-stimulated insulin secretion (GSIS) from islets of WT and 2A AR KO mice was analyzed in a perifusion apparatus following exposure to different levels of glucose in the absence or presence of an 2 agonist. Insulin secretion by islets isolated from WT mice was stable for four repetitive stimulations with 16·8 mM glucose, and addition of the 2 antagonist, yohimbine, had no effect (Fig. 1A) . As shown in Fig. 1B , addition of 5 10 9 M UK 14 304, an 2 AR agonist, caused a significant decrease in glucose-stimulated insulin secretion. The 2A AR antagonist, yohimbine, partially reversed this response, presumably by preventing agonist re-binding to the 2A AR during the UK 14 304 wash-out. The recovery of glucose-stimulated insulin release during perifusion with yohimbine confirms that the suppression of insulin release during the UK 14 304 perifusion was due to receptor-mediated regulation, and not simply a decline in the responsiveness of the islets. In preliminary studies, we determined that the inhibition of insulin release by UK 14 304 is concentrationdependent, with complete suppression at 1 10 7 M. We used the same approach to analyze the insulin secretion from islets isolated from 2A AR KO mice ( Fig. 1C and D) . First, it was noted that 2A AR KO islets secreted less insulin in response to 16·8 mM glucose. Peterhoff et al. (2003) showed that 2A AR islets secreted less insulin in response to 16·8 mM glucose plus forskolin and IBMX, but they were unable to demonstrate normal glucose-stimulated insulin secretion (16·8 mM glucose) in freshly isolated normal or KO islets. Our results demonstrate that islets from 2A AR KO mice can respond to glucose stimulation, but with reduced insulin output.
Inhibition of glucose-stimulated insulin release by the 2 AR agonist UK 14 304 is eliminated in 2A AR KO islets
Since the amount of insulin release by WT islets in response to 16·8 mM glucose (45·2 ng/ml) is significantly greater than that released from 2A AR KO islets (18·6 ng/ml), we measured pancreatic insulin content in 2A AR WT and KO mice. The pancreatic insulin content in 2A AR KO mice (9·60 2·07 µg/100 mg pancreas) was not significantly different from the insulin content in the pancreas of WT mice (11·03 1·61 µg/ 100 mg pancreas). We measured total pancreatic insulin to avoid any ascertainment bias due to variation in islet size. Our results measuring pancreatic insulin content corroborate data of Peterhoff et al. (2003) who measured islet insulin content, and indicate that a difference in insulin content is not the reason that 2A AR KO mice secrete less insulin in response to glucose stimulation.
Inhibition of glucose-stimulated insulin release by the 2 AR agonist UK 14 304 is eliminated in 2A AR KO islets
In contrast to WT islets, UK 14 304 did not inhibit insulin secretion from islets isolated from 2A AR KO mice. Analysis of data from multiple experiments (Fig. 1E) revealed that whereas 5 10 9 M UK 14 304 caused 77·6% inhibition of insulin release from islets harvested from WT mice, there is no significant 2 agonist-evoked decrease in insulin release from islets harvested from 2A AR KO mice. These results demonstrate that the effects of the 2A AR agonist, UK 14 304, on WT islets are mediated via the 2A AR subtype.
Differential protein expression in islets from WT and

2A AR KO mice
One goal of these studies was to use a proteomics strategy to explore differences in protein profiles in islets from WT or 2A AR mice since this might reveal novel downstream targets of 2A AR signaling or pleiotropic consequences of perturbed 2A AR expression. Initially, we explored whether 2 AR agonist treatment of islets from WT mice led to acute changes in protein expression, and/or detectable post-translational modification, as assessed by two-dimensional difference gel electrophoresis (2D-DIGE). Pancreatic islets isolated from WT mice were treated for 5 min with 10 6 M dexmedetomidine, a 2A AR agonist, and the proteomic variation compared between treated and control islets. Five differentially expressed spots (corresponding to cytokeratin 8 and 18; claude B protease inhibitor; amylase; and chymotrypsinogen B precursor) were identified in this comparative proteomic approach, but all had a small variation (between 1·24-fold and 1·53-fold). These findings indicate that if acute changes in protein phosphorylation or other post-translational modifications occur, they are not detectable via this methodology for this particular biological system under the conditions of our experiments. Consequently, we did not pursue this strategy further.
2A AR KO mice
The 2D-DIGE approach was next used to investigate the proteomic variation between pancreatic islets from WT and 2A AR KO mice. As shown schematically in Fig. 2A , three sequential steps comprised this strategy: 1) Islet samples from WT mice were labeled by Cy3, and mixed with Cy5-labeled islets from 2A AR KO mice. 2) Proteins in the mixed sample were separated by 2D-DIGE. The similar mass (500 Da), monoprotonation, pKa, and charge of the dyes on thé -amino group of lysines they substitute ensures the same comigration patterns of proteins labeled by either one of the dyes (Alban et al. 2003) .
2A AR KO mice
3) The resulting gel was scanned separately for detection of the Cy3 image by a 532 nm laser and an emission filter of 580 nm and the Cy5 image by a 633 nm laser and an emission filter of 670 nm. The generated images were analyzed by DeCyder software to detect and quantify the changes in individual protein spots on the gel. The intensity of the signal over a large dynamic (>10 4 ) quantity range (Unlu et al. 1997) relates directly to the amount of protein labeled, because the low dye:protein ratio (minimal labeling) ensures that a single fluorophor-molecule labels a single protein molecule (Tonge et al. 2001) . Each islet sample consisted of a pool of 10 independent islet isolations, thereby limiting non-biological variation introduced during islet isolation.
2A AR KO mice
The Cy3 (WT) and Cy5 ( 2A AR KO) spot maps comparing islet extracts from WT vs 2A AR KO mice were highly reproducible over three separate islet pools and 2D gel analyses. For each gel, 1500 spots were resolved over a pH range of 4 to 7 (Fig. 2B) . The plot shown in Fig. 2C displays the analysis of the abundance ratios for every spot-pair generated from both images using DeCyder software. The overall variation in abundance ratios was modeled by a normal distribution, from which two S.D. of the mean were calculated and used for the 95 th percentile confidence level. We found that 25 protein spots were significantly increased in abundance in islets from 2A AR KO mice (Fig. 3) . These 25 proteins were targeted for subsequent protein identification by mass spectrometry and database interrogation. For the proteins described in Table 1 , the approximate fold change shown in the table is calculated from a sample obtained by pooling extracts from islets Figure 1 Islet perifusion analysis of glucose-stimulated insulin secretion (GSIS) from islets isolated from wild type (WT) and 2A AR knockout (KO) mice. Forty islets were used for each assay. Perifusion columns were immerged in a 37°C water bath and perifusion was performed at a flow rate of 1 ml/min as described in Materials and methods. The x-axis shows the time of perifusion and the y-axis shows the insulin concentration in each perifusion sample (note different y-axis scale in panels A, B and C, D). The bar at the top of each graph shows the glucose concentration in the perifusate (open bar=2·8 mM glucose; black bar=16·8 mM glucose). (A) Insulin secretion from one representative experiment in response to repetitive exposure to 16·8 mM glucose in islets pooled from four WT mice. (B) Insulin secretion in response to treatment of 16·8 mM glucose and 5×10 −9 M UK14 304 in islets from WT mice. (C) Insulin secretion in response to repetitive exposure of 16·8 mM glucose in 2A AR KO mice islets; mean±S.E., n=3. Islets studied in three separate experiments using islets pooled from four mice in each experiment. (D) Insulin secretion in response to 16·8 mM glucose ±5×10 −9 M UK14 304 in 2A AR KO islets; mean±S.E., n=3. Islets studied in three separate experiments using islets pooled from four mice in each experiment. (E) Inhibition of GSIS by 5×10 −9 M UK14 304 in WT and 2A AR KO islets. Data from multiple experiments performed as in panel B (WT) and D ( 2A AR KO) were analyzed to estimate the amount of insulin release evoked by 16·8 mM glucose ±UK 14 304 from three independent experiments for each genotype. Preliminary studies had shown that the effect of UK 14 304 was dose-dependent with complete suppression occurring at 1×10 −7 M UK14 304. SigmaPlot 8·0 (SPSS Inc., Chicago, IL, USA) was used to quantify the area under the curve for 16·8 mM glucose-evoked insulin release. The data for stimulation by 16·8 mM glucose represent the mean of the first two high-glucose stimulations in a given experiment. The data for 16·8 mM glucose+5nM UK 14 304 are the area under the curve for this treatment in each experiment.
2A AR KO mice
from each of 10 mice of each (WT, KO) genotype to normalize for mouse to mouse and islet to islet variability. Table 1 summarizes the identification of these 'up-regulated' proteins -many of which are involved in lipid metabolism, serine biosynthesis, transcriptional regulation, proteolysis and peptidolysis, and peroxidase reactions, that are characteristic of metabolic pathways in endocrine secretory cells. For example, carboxypeptidase B1 protein, critical for the processing of insulin protein in pancreatic islets (Kemmler et al. 1971) , was increased in the extracts of islets from the 2A AR KO mice.
2A AR KO mice
Although most of the protein spots that differed between WT and 2A AR KO mice reflected an increase in intensity, the intensity of spot 9a was decreased in islets obtained from 2A AR KO mice (Fig. 2B and D) . A reciprocal increase in expression in spot 9b was observed in islets from 2A AR KO mice. The similar molecular weight of the two spots but dissimilar pixel intensity suggested the possibility that spot 9b might be a post-translational modification of the protein migrating as spot 9a. Mass spectrometry analysis of spot 9a from WT mice demonstrated that this spot corresponds to a published amino acid sequence with genbank accession number NP_035924 (gi:6753204, 23 (Fig. 4) . This difference in the peptide mass maps was sufficient to distinguish these isoforms during database interrogation.
2A AR KO mice
Several possibilities might explain the differential expression of Bpnt1 isoforms in WT vs 2A AR KO mice, such as regulated expression of one vs another allele of Bpnt1 in WT vs 2A AR KO mice. Another possibility was that the detected isoforms were indicative of genetic variation introduced during manipulation of the mouse genome by homologous recombination. The reference cDNA sequences encoding protein NP_035924 (accession number NM_011794 (Spiegelberg et al. 1999) ) and AAH11036 (accession number BC011036 (Strausberg et al. 2002) ) suggested a strategy to identify the genetic origin of Bpnt1 in islets from 2A -AR KO mice (Fig. 5) . The 2A AR KO mice were generated by implanting correctly targeted R1 ES cells into eight cell FVB/N embryos, followed by transferring the embryos into pseudopregnant mice (Altman et al. 1999) . The chimeric mice were backcrossed with C57BL6 mice six times, so that the R1 ES cell genome background was effectively diluted to 1·5625% of its original. To determine whether the differentially expressed bisphosphate 3 -nucleotidase 1 isoforms in WT vs 2A AR KO mice resulted from strain difference carried by R1 ES cell genome background, we isolated the genomic DNA from WT mice, 2A AR KO mice, and R1 ES cell line. A region of Bpnt1 locus diagnostic for NM_011794 (WT) and the BC011036 ( 2A AR KO) Bpnt1 isoforms was amplified by PCR, and an restriction fragment length polymorphism (RFLP) strategy was used to analyze the bisphosphate 3 -nucleotidase 1 isoform in each source.
2A AR KO mice
As shown in Fig. 6A , two primers flanking the coding sequence of exon 8 were exploited to amplify a 401 bp genomic DNA fragment, which includes exon 8 and part of the intron sequences on both sides. Analysis of the two Table 1 . This gel represents Sypro Ruby staining of the gel shown in Fig. 2 . The designated spots were excised, hydrolyzed by trypsin, and analyzed by MALDI-TOF and/or ms-ms as described in Materials and methods.
2A AR KO mice
published cDNA sequences encoding each isoform revealed a differential digestion pattern by the restriction enzyme Hha I. In WT mice, the amplified 401 bp fragment was digested into two fragments of 89 bp and 312 bp, with the restriction recognition site at position 561 of the published sequence NM_011794. In 2A AR KO mice, digestion generated two fragments of 166 bp and 235 bp, with the restriction recognition site at position 634 of the reported sequence BC011036 (Fig. 6A) .
2A AR KO mice
To further confirm the sequence of the PCR product isolated from WT mice, 2A AR KO mice, and R1 ES cells, the amplified 401 bp fragment (Fig. 6B) was submitted for sequencing. The same purified DNA sample was used for sequencing and Hha I digestion. All four nucleotide variations detected in genomic samples from 2A AR KO mice are identical to those detected in the R1 ES cell line (labeled sites in Fig. 6C ), suggesting that the 2A AR KO mice carry the genomic background from R1 stem cell line at both alleles at the bisphosphate 3 -nucleotidase 1 locus. If 2A AR KO mice are heterozygous at Bpnt1 site, carrying one copy from each isoform, the restriction digest pattern should display four bands. If the 2A AR KO mice are homozygous for either isoform, the predicted digestion pattern should show two bands, representing each isoform (Fig. 6D) . The observation that the Hha I enzyme digestion pattern from our 2A AR KO mice is the same as that from the R1 stem cell line indicates that the mice are homozygous for the Bpnt1 gene encoding the enzyme isoform from R1 stem cell line (Fig. 6E) .
Discussion
During the course of our studies, Peterhoff et al., (2003) using mice null for the 2A AR, reported a principal role for the 2A AR subtype in membrane hyperpolarization, suppression of cAMP accumulation and attenuation of cAMP-evoked GSIS ex vivo. Our studies corroborate and extend those findings by demonstrating the role of the 2A AR in suppressing insulin release stimulated by the endogenous secretagogue, glucose. The data from Peterhoff et al. (2003) in 2A / 2C AR double knockout mice also suggest an ancillary role of the 2C AR subtype. Why suppression of insulin release in response to UK 14 304 is entirely eliminated in 2A AR KO mice in our studies, whereas epinephrine-inhibited insulin release was reduced only 70% in their studies of Peterhoff et al. (2003) is not clear, although we suspect that the higher affinity of UK 14 304 compared with epinephrine at 2A AR means that the 2A ARs are virtually continuously occupied by the 2 agonist UK 14 304 during the course of our perifusion studies, accounting for the 100% suppression of insulin release observed. The grey dotted line corresponds to the sequence of the Bpnt1 isoform expressed in islets isolated from wild type (WT) mice, corresponding to Genbank accession number NM_011794; the blue dashed line indicates the sequence of the Bpnt1 isoform identified in islets from 2A AR knockout (KO) mice and the R1 ES cell line, corresponding to Genbank accession number BC011036. Encoded amino acid sequences also are shown. Bpnt1 nucleotide changes which alter the amino acid sequence (561C-556T; 639A-634 G) are numbered and labeled with red letters; two additional nucleotide differences encoded by these two Bpnt1 isoforms (625C-621T; 649A-644C) do not alter the amino acid sequence and are labeled in green letters. Nucleotides are numbered according to the published cDNA sequence of each isoform. Start sites of each of the PCR primers to amplify the sequence diagnostic for each Bpnt1 isoforms are indicated as brown arrows. Primer sequences are in orange letters. ➀ and ➁ are Hha 1 restrictive enzyme sites, further described in Fig. 6 .
Discussion
Using a 2D-DIGE proteomic approach to analyze the steady state protein expression in WT vs KO 2A AR mice, we found several significant changes. MALDI mass spectrometry enabled successful matching of all 2D spots with known proteins in the database, which enabled us to utilize the functional information linked in the mouse genome database to analyze all proteins identified. Among these proteins, of particular interest to us were those involved in insulin processing, biosynthesis and/or regulated secretory release. Our study also revealed significant changes in content for proteins involved in biosynthesis, enzyme secretion, and other pancreatic functions (Table 1) . These changes may be due to loss of 2A AR regulation in the pancreas or other 2A AR-expressing tissues during development and in the adult animal, but may also be consequences of resultant changes in insulin and glucose homeostasis in 2A AR KO mice. One of those proteins increased significantly in 2A AR KO mice was carboxypeptidase B1 (Cpb1). Steiner and colleagues have reported that carboxypeptidase B as well as trypsin is involved in the conversion from proinsulin to insulin (Kemmler et al. 1971) . Indeed, the level of all eight spots corresponding to carboxypeptidase B1 (5a-h) increased significantly from 3·86-fold to 6·41-fold in a reproducible manner. Although speculative, the increase in an enzyme involved in insulin processing in 2A AR KO mice may represent an appropriate compensatory mechanism to the loss of tonic suppression of insulin release (thus leading to tonic enhancement of insulin release) in the 2A AR KO mice.
Discussion
The observation that Bpnt1 differences in WT vs KO mice resulted from the genetic contribution of ES cells in the 2A AR KO mice, rather than 2A AR regulation of gene expression suggests that inclusion of studies to profile ES cell vs WT animals is necessary to identify proteomic changes not due to post-translational modification in genetically engineered mouse lines.
Discussion
Overall, our findings confirm the role of the 2A AR subtype in the suppression of insulin secretion from pancreatic islets in response to glucose, the endogenous secretagogue, and in more pleiotropic changes in enzymes and pathways related to insulin processing either due to changes in pancreatic pathways or to changes in other 2A AR-expressing tissues, including regulatory pathways in the brain. Restoring these contributions will require the future study of mice where the expression of the 2A AR can be controlled in a tissue-specific and time-dependent fashion.

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