Expression cloning of a reserpine-sensitive vesicular monoamine transporter.

A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent Km of 1.3 microM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H(+)-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules.     

Differential quantal release of histamine and 5-hydroxytryptamine from mast cells of vesicular monoamine transporter 2 knockout mice

The recent availability of mice lacking the neuronal form of the vesicular monoamine transporter 2 (VMAT2) affords the opportunity to study its roles in storage and release. Carbon fiber microelectrodes were used to measure individual secretory events of histamine and 5-hydroxytryptamine (5-HT) from VMAT2-expressing mast cells as a model system for quantal release. VMAT2 is indispensable for monoamine storage because mast cells from homozygous (VMAT2(-/-)) mice, while undergoing granule-cell fusion, do not release monoamines. Cells from heterozygous animals (VMAT2(+/-)) secrete lower amounts of monoamine per granule than cells from wild-type controls. Investigation of corelease of histamine and 5-HT from granules in VMAT2(+/-) cells revealed 5-HT quantal size was reduced more than that of histamine. Thus, although vesicular transport is the limiting factor determining quantal size of 5-HT and histamine release, intragranular association with the heparin matrix also plays a significant role.     

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.     

Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo

Disruption of neurotransmitter vesicle dynamics (transport, capacity, release) has been implicated in a variety of neurodegenerative and neuropsychiatric conditions. Here, we report a novel mouse model of enhanced vesicular function via bacterial artificial chromosome (BAC)-mediated overexpression of the vesicular monoamine transporter 2 (VMAT2; Slc18a2). A twofold increase in vesicular transport enhances the vesicular capacity for dopamine (56%), dopamine vesicle volume (33%), and basal tissue dopamine levels (21%) in the mouse striatum. The elevated vesicular capacity leads to an increase in stimulated dopamine release (84%) and extracellular dopamine levels (44%). VMAT2-overexpressing mice show improved outcomes on anxiety and depressive-like behaviors and increased basal locomotor activity (41%). Finally, these mice exhibit significant protection from neurotoxic insult by the dopaminergic toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), as measured by reduced dopamine terminal damage and substantia nigra pars compacta cell loss. The increased release of dopamine and neuroprotection from MPTP toxicity in the VMAT2-overexpressing mice suggest that interventions aimed at enhancing vesicular capacity may be of therapeutic benefit in Parkinson disease.Faulty monoamine neurotransmission is characteristic of many disorders, including Parkinson disease, depression, dystonia, attention deficit hyperactivity disorder, schizophrenia, addiction, and Huntington disease (1–7). Several strategies have been used to enhance monoamine signaling: administration of precursors to increase synthesis, inhibition of enzymes to prevent metabolism/degradation, inhibition of plasma membrane transporters to increase synaptic lifespan, and administration of receptor agonists to directly activate postsynaptic targets. However, these therapies fail to preserve many, if not all, of the critical aspects of chemical neurotransmission: normal transmitter synthesis, activity-dependent transmitter release and receptor activation, and receptor recovery following signal termination both by transmitter uptake and metabolism. Thus, these approaches often produce deleterious side effects or lose efficacy over time.Increasing the neurotransmitter content in the synaptic vesicle may represent a therapeutic approach capable of increasing the release of monoamines without the aforementioned adverse effects. The vesicular monoamine transporter 2 (VMAT2, SLC18A2) is responsible for the packaging of neurotransmitter into vesicles for subsequent release from monoaminergic neurons. VMAT2 is an H⁺-ATPase antiporter, which uses the vesicular electrochemical gradient to drive the packaging of cytosolic transmitter into small synaptic and dense core vesicles (8–10). VMAT2 is also essential for survival of dopamine neurons as cytosolic dopamine is neurotoxic (11, 12). By sequestering intracellular dopamine into vesicles, VMAT2 prevents cytosolic dopamine accumulation and its subsequent conversion to neurotoxic species (13–17). Thus, VMAT2 serves two primary functions: to mediate monoamine neurotransmission and to counteract intracellular toxicity.Previous data clearly show that disruption of VMAT2 function produces adverse effects. Pharmacological VMAT2 inhibition by reserpine or tetrabenazine results in monoamine depletion and negative behavioral consequences, including akinesia and depressive behaviors (18–20). Genetic reduction of VMAT2 in mice also causes depletion of dopamine, norepinephrine, and serotonin and progressive neurodegeneration in multiple monoaminergic regions (21–25). Similarly, a VMAT2 mutation in humans that dramatically reduces vesicular function was recently linked to an infantile parkinsonism-like condition with symptoms caused by deficits in all of the monoamines (26). SNPs in the VMAT2 gene have also been associated with neurocognitive function in relatives of patients with schizophrenia and even in posttraumatic stress disorder (27, 28). Interestingly, haplotypes within the VMAT2 promoter region that increase VMAT2 expression have been associated with a decreased risk of Parkinson disease (29, 30). Although the detrimental effects of reduced VMAT2 function are recognized, our understanding of the potential benefits of increased VMAT2 function in vivo has been limited to a Drosophila model (31–33). Thus, we generated VMAT2-overexpressing mice using a bacterial artificial chromosome (BAC) to determine whether increased vesicular packaging could provide an elevation of monoamine output in a mammalian system. We report here that VMAT2-overexpressing mice (VMAT2-HI) have increased vesicle capacity, increased synaptic dopamine release, improved outcomes on anxiety-like and depressive-like behaviors, and reduced vulnerability to toxic insult by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These data demonstrate that the manipulation of vesicular capacity is capable of providing a sustained enhancement of the dopamine system and suggest that the vesicle is a viable therapeutic target for numerous monoamine-deficient disorders.     

Role of vesicular monoamine transporter type 2 in rodent insulin secretion and glucose metabolism revealed by its specific antagonist tetrabenazine

Despite different embryological origins, islet beta-cells and neurons share the expression of many genes and display multiple functional similarities. One shared gene product, vesicular monoamine transporter type 2 (VMAT2, also known as SLC18A2), is highly expressed in human beta-cells relative to other cells in the endocrine and exocrine pancreas. Recent reports suggest that the monoamine dopamine is an important paracrine and/or autocrine regulator of insulin release by beta-cells. Given the important role of VMAT2 in the economy of monoamines such as dopamine, we investigated the possible role of VMAT2 in insulin secretion and glucose metabolism. Using a VMAT2-specific antagonist, tetrabenazine (TBZ), we studied glucose homeostasis, insulin secretion both in vivo and ex vivo in cultures of purified rodent islets. During intraperitoneal glucose tolerance tests, control rats showed increased serum insulin concentrations and smaller glucose excursions relative to controls after a single intravenous dose of TBZ. One hour following TBZ administration we observed a significant depletion of total pancreas dopamine. Correspondingly, exogenous L-3,4-dihydroxyphenylalanine reversed the effects of TBZ on glucose clearance in vivo. In in vitro studies of rat islets, a significantly enhanced glucose-dependent insulin secretion was observed in the presence of dihydrotetrabenazine, the active metabolite of TBZ. Together, these data suggest that VMAT2 regulates in vivo glucose homeostasis and insulin production, most likely via its role in vesicular transport and storage of monoamines in beta-cells.     

Gastrin affects enzyme activity and gene expression in the aging rat pancreas.

The structural and functional properties of the pancreas are known to be affected by a number of hormones, particularly those of the gastrin-CCK family, yet little is known about the responsiveness of the pancreas to gastrin-CCK peptides during the latter stages of life. The present investigation examined the changes in pancreatic growth, the activity, and the steady-state mRNA levels of some of the digestive enzymes during advancing age and after administration of gastrin. Groups of 3-, 6-, 12-, and 16-month-old male Fischer-344 rats were infused (osmotic minipump) with either gastrin G-17 (250 ng/kg/h) or saline (controls) for 14 days. In control pancreas, aging resulted in slight progressive reduction in pancreatic DNA, RNA, and protein concentrations. This decrease was markedly enhanced by gastrin treatment in 16-month-old rats. Pancreatic amylase and trypsin (TRP) activities in these animals were also slightly decreased with aging, whereas the steady-state mRNA levels of both enzymes were significantly higher in 16-month-old rats than in their 3-month-old counterparts. However, in 16-month-old rats, the steady-state mRNA levels of amylase and TRP were significantly reduced after gastrin administration, when compared with the corresponding controls. Chymotrypsin (CHY) activity in the pancreas remained essentially unchanged between 3- and 12-month-old rats, but in 16-month-old animals it was markedly decreased. CHY activity was further reduced by gastrin treatment only in the 16-month-old group.(ABSTRACT TRUNCATED AT 250 WORDS)     

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.     

Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models

Aims/hypothesis

Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans.

Methods

We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2.

Results

The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species.

Conclusions/interpretation

Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a ‘null’ model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas.     

Molecular cloning of a putative vesicular transporter for acetylcholine.

Classical neurotransmitters such as acetylcholine (ACh) require transport into synaptic vesicles for regulated exocytotic release. The Caenorhabditis elegans gene unc-17 encodes a protein with homology to mammalian transporters that concentrate monoamine neurotransmitters into synaptic vesicles. Mutations in unc-17 protect against organophosphorus toxicity, indicating a role in cholinergic neurotransmission. Using the relationship of unc-17 to the vesicular amine transporters, we first isolated a related sequence from the electric ray Torpedo californica [Torpedo vesicular ACh transporter (TorVAChT)] that is expressed by the electric lobe but not by peripheral tissues. Using the relationship of the Torpedo sequence to unc-17, we then isolated the cDNA for a rat homologue (rVAChT). Northern blot analysis shows expression of these sequences in the basal forebrain, basal ganglia, and spinal cord but not cerebellum or peripheral tissues. In situ hybridization shows expression of rVAChT mRNA in all cholinergic cell groups, including those in the basal forebrain, brainstem, and spinal cord that previously have been shown to express choline acetyltransferase mRNA. The human VAChT gene also localizes to chromosome 10 near the gene for choline acetyltransferase. Taken together, these observations support a role for rVAChT in vesicular ACh transport and indicate its potential as a novel marker for cholinergic neurons.     

MicroRNA-373 induces expression of genes with complementary promoter sequences

Recent studies have shown that microRNA (miRNA) regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report new evidence in which miRNA may also function to induce gene expression. By scanning gene promoters in silico for sequences complementary to known miRNAs, we identified a putative miR-373 target site in the promoter of E-cadherin. Transfection of miR-373 and its precursor hairpin RNA (pre-miR-373) into PC-3 cells readily induced E-cadherin expression. Knockdown experiments confirmed that induction of E-cadherin by pre-miR-373 required the miRNA maturation protein Dicer. Further analysis revealed that cold-shock domain-containing protein C2 (CSDC2), which possesses a putative miR-373 target site within its promoter, was also readily induced in response to miR-373 and pre-miR-373. Furthermore, enrichment of RNA polymerase II was detected at both E-cadherin and CSDC2 promoters after miR-373 transfection. Mismatch mutations to miR-373 indicated that gene induction was specific to the miR-373 sequence. Transfection of promoter-specific dsRNAs revealed that the concurrent induction of E-cadherin and CSDC2 by miR-373 required the miRNA target sites in both promoters. In conclusion, we have identified a miRNA that targets promoter sequences and induces gene expression. These findings reveal a new mode by which miRNAs may regulate gene expression.     

Gastrin activity along the gastrointestinal tracts of some ruminants and the donkey.

Using the Blair et al. (1961) gastrin extraction method, attempts were made to extract gastrin from the fresh mucosae of the various sections of the gastrointestinal tracts of cattle, goat, sheep, male and female donkeys. Gastrin activity was only detected in the pyloric antral and duodenal extracts in all the animals. The crude pyloric and duodenal extracts expressed in milligrams of wet weight of the mucosa were 28.13 ± 5.0 and 24.05 ± 6.52, respectively for cattle; 18.75 ± 4.39 and 15.65 ± 4.25 for sheep; 27.43 ± 5.04 and 18.50 ± 3.5 for goat; 24.21 ± 4.81 and 22.15 ± 3.25 for male donkey; 18.09 ± 1.34 and 15.52 ± 5.2 for female donkey. The differences were found to be significantly higher in the pyloric antra than in the duodenal mucosa in cattle, sheep, and goat (P < 0.002 > 0.01) while nonsignificant in both the male and female donkeys (P > 0.10). The saline solutions of the gastrin extracts from the pyloric mucosa contained more gastrin than the corresponding solutions of the duodenal extracts in all the animals. The differences in gastrin activity expressed in nanograms synthetic human gastrin I (SHG I) per milligrams of extracts of the pyloric and duodenal mucosae were significant in all the animals (P < 0.001).     

Identification and characterization of a novel isoform of the vesicular gamma-aminobutyric acid transporter with glucose-regulated expression in rat islets

Pancreatic islets are unique outside the nervous system in that they contain high levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), synthesized by the enzyme glutamic acid decarboxylase (GAD). Since the role that GABA plays in the islet and the mechanisms whereby the two major GAD isoforms (GAD65 and GAD67) function as diabetes-associated autoantigens are unknown, continued characterization of the islet GAD-GABA system is important. We previously demonstrated that the GABA and glycine transporter vesicular inhibitory amino acid transporter (VIAAT also known as VGAT) is present in rat islets. Here we identify a novel 52 kDa variant of VIAAT in rat islets: VIAAT-52 (V52). V52 is an amino-terminally truncated form of VIAAT (V57) that likely results from utilization of a downstream start site of translation. V57 and V52 display different patterns of post-translational modification and cellular expression. Our results have indicated that islet content of V52, but not V57, is responsive to changes in glucose concentration and other extracellular conditions. VIAAT is expressed in the islet alpha cells, but there have been conflicting findings regarding the presence of VIAAT in the beta cells. Here we have also provided additional evidence for the presence of VIAAT in islet beta cells and show that the beta cell line INS-1 expresses V57. V52 may be better adapted than V57 to the unique rat alpha cell GAD-GABA system, which lacks GAD65 and in which VIAAT traffics to secretory granules rather than just to synaptic microvesicles.     

Diabetes alters the expression and activity of the human placental GLUT1 glucose transporter

This study was designed to investigate the effects of maternal diabetes on glucose transporter expression and glucose transport activity in the human placenta. Syncytiotrophoblast microvillous and basal membranes were prepared from placental tissue obtained at term from pregestational diabetics (White class B) and gestational diabetics controlled either by diet alone (class A1) or by diet and insulin (class A2). These membranes were used to measure GLUT1 glucose transporter expression and D-glucose transport activity. Diabetic groups showed no differences in placental weights or neonatal birth weights compared to controls, although 8 of 25 diabetic fetuses were macrosomic. Glycemic control in the diabetics at term, as assessed by maternal glycosylated hemoglobin, was within normal limits. Basal membrane GLUT1 density was about 2-fold higher in all diabetic groups compared to that in controls, as measured by immunoblotting, whereas no changes were found for the microvillous membranes. D-Glucose uptake across the basal membrane was increased by 40% in the diabetic groups; no changes were observed for the microvillous membrane. These results demonstrate that diabetes causes an increase in basal membrane GLUT1 expression and activity that persists despite a lack of evidence for current or recent maternal hyperglycemia. This suggests the potential for an extended increase in transplacental glucose flux in the absence of maternal hyperglycemia, which may contribute to fetal macrosomia and the other consequences of diabetic pregnancy.     

Functional and molecular characterization of a muscarinic receptor type and evidence for expression of choline-acetyltransferase and vesicular acetylcholine transporter in human granulosa-luteal cells.

Previously, we provided evidence for the presence of a class of muscarinic receptors on human luteinized granulosa cells (human GC) that is linked to transient increases in intracellular free calcium levels, but not to steroid production. The precise nature of the receptor is not known, and neither its function nor the source of its natural ligand acetylcholine (ACh) is clear. To address these issues we used RT-PCR approaches and isolated complementary DNAs corresponding to the M1 receptor subtype from reverse transcribed human GC messenger ribonucleic acids. M1 receptors were further shown by immunocytochemistry, using a M1 receptor antiserum. Single cell calcium measurements showed that the M1 receptor was functionally active and linked to acute increases in intracellular free calcium, as the M1 receptor specific antagonist pirenzepine blocked the Ca2+-mobilizing effect of oxotremorine M (a muscarinic agonist). An unexpected consequence of M1 receptor activation was evidenced by the ability of muscarinic agonists to stimulate the proliferation of human GC within 24 h. In vivo, ACh, the natural ligand of these receptors is thought to be contained in cholinergic nerve fibers innervating the ovary. Surprisingly, the prerequisite for the synthesis of ACh, the enzyme choline-acetyltransferase (ChAT), is also expressed by human GC, as shown by Western blotting and immunocytochemistry. In addition, these cells express another marker for ACh synthesis, namely the gene for the vesicular acetylcholine transporter, as evidenced by RT-PCR cloning, Western blotting, and immunocytochemistry. In conclusion, our data identify the M1 receptor in human GC and point to a novel, trophic role of the neurotransmitter ACh. Furthermore, the presence of the prerequisites of ACh synthesis in human GC indicate that an autocrine/paracrine regulatory loop also exists in the in vivo counterparts of these cells in the ovary, i.e. in the cells of the preovulatory follicle and/or of the young corpus luteum.     

Vesicular monoamine transporter,type 2 (vmat2) expression as it compares to insulin and pancreatic polypeptide in the head,body and tail of the human pancreas

The vesicular monoamine transporter, type 2 (VMAT2) is responsible for sequestering monoamine neurotransmitters into exocytic vesicles in neurons, enterochromaffin-like cells of the stomach and cells arising from the common myeloid progenitor. VMAT2 is also present in the pancreas and is expressed by insulin producing β cells, but not by glucagon or somatostatin expressing islet cells. Positron emission tomography (PET) targeting of VMAT2 is currently being evaluated as a non-invasive tool to measure β cell mass (BCM) in living humans. In recent trials, PET measurements of VMAT2 in the pancreas overestimated BCM in type 1 diabetes (T1D) patients predicted to have little to no BCM by metabolic measures. Recently, tissue immunohistochemistry studies suggested that VMAT2 staining may also co-localize with pancreatic polypeptide (PP) staining cells in pancreas tissue, but these studies were not quantitative. In this report, we evaluated VMAT2 specificity for β cells in sub-regions of the human pancreas using antibodies targeting VMAT2, insulin and PP by double-label immunofluorescence. Immunostaining for VMAT2 and insulin demonstrated 89 ± 8% overlap in the body and tail of the pancreas. However, 44 ± 12% and 53 ± 15% of VMAT2 cells co-stained with PP- and insulin-staining cells, respectively in the pancreatic head. Significant co-staining for VMAT2 and PP cells in the head of the pancreas may partly explain the apparent overestimation of BCM in T1D by PET. Specific targeting of the pancreatic body and tail using VMAT2 PET scanning may reflect BCM more accurately.     

Vesicular monoamine transporter,type 2 (vmat2) expression as it compares to insulin and pancreatic polypeptide in the head,body and tail of the human pancreas

The vesicular monoamine transporter, type 2 (VMAT2) is responsible for sequestering monoamine neurotransmitters into exocytic vesicles in neurons, enterochromaffin-like cells of the stomach and cells arising from the common myeloid progenitor. VMAT2 is also present in the pancreas and is expressed by insulin producing β cells, but not by glucagon or somatostatin expressing islet cells. Positron emission tomography (PET) targeting of VMAT2 is currently being evaluated as a non-invasive tool to measure β cell mass (BCM) in living humans. In recent trials, PET measurements of VMAT2 in the pancreas overestimated BCM in type 1 diabetes (T1D) patients predicted to have little to no BCM by metabolic measures. Recently, tissue immunohistochemistry studies suggested that VMAT2 staining may also co-localize with pancreatic polypeptide (PP) staining cells in pancreas tissue, but these studies were not quantitative. In this report, we evaluated VMAT2 specificity for β cells in sub-regions of the human pancreas using antibodies targeting VMAT2, insulin and PP by double-label immunofluorescence. Immunostaining for VMAT2 and insulin demonstrated 89 ± 8% overlap in the body and tail of the pancreas. However, 44 ± 12% and 53 ± 15% of VMAT2 cells co-stained with PP- and insulin-staining cells, respectively in the pancreatic head. Significant co-staining for VMAT2 and PP cells in the head of the pancreas may partly explain the apparent overestimation of BCM in T1D by PET. Specific targeting of the pancreatic body and tail using VMAT2 PET scanning may reflect BCM more accurately.