Antonella Maffei

Longitudinal noninvasive PET-based β cell mass estimates in a spontaneous diabetes rat model

Diabetes results from an absolute or relative reduction in pancreatic beta cell mass (BCM) leading to insufficient insulin secretion and hyperglycemia. Measurement of insulin secretory capacity is currently used as a surrogate measure of BCM. However, serum insulin concentrations provide an imprecise index of BCM, and no reliable noninvasive measure of BCM is currently available. Type 2 vesicular monoamine transporters (VMAT2) are expressed in human islet beta cells, as well as in tissues of the CNS. [11C]Dihydrotetrabenazine ([11C]DTBZ) binds specifically to VMAT2 and is a radioligand currently used in clinical imaging of the brain. Here we report the use of [11C]DTBZ to estimate BCM in a rodent model of spontaneous type 1 diabetes (the BB-DP rat). In longitudinal PET studies of the BB-DP rat, we found a significant decline in pancreatic uptake of [11C]DTBZ that anticipated the loss of glycemic control. Based on comparison of standardized uptake values (SUVs) of [11C]DTBZ and blood glucose concentrations, loss of more than 65% of the original SUV correlated significantly with the development of persistent hyperglycemia. These studies suggest that PET-based quantitation of VMAT2 receptors provides a noninvasive measurement of BCM that could be used to study the pathogenesis of diabetes and to monitor therapeutic interventions.

11C-Dihydrotetrabenazine PET of the Pancreas in Subjects with Long-Standing Type 1 Diabetes and in Healthy Controls

Type 2 vesicular monoamine transporter (VMAT2), found in the brain, is also expressed by β-cells of the pancreas in association with insulin. Preclinical experiments suggested that 11C-dihydrotetrabenazine PET–measured VMAT2 binding might serve as a biomarker of β-cell mass. We evaluated the feasibility of 11C-dihydrotetrabenazine PET quantification of pancreatic VMAT2 binding in healthy subjects and patients with long-standing type 1 diabetes. Methods: 11C-Dihydrotetrabenazine PET was performed on 6 patients and 9 controls. VMAT2 binding potential (BPND) was estimated voxelwise by using the renal cortex as reference tissue. As an index of total pancreatic VMAT2, the functional binding capacity (the sum of voxel BPND × voxel volume) was calculated. Pancreatic BPND, functional binding capacity, and stimulated insulin secretion measurements were compared between groups. Results: The pancreatic mean BPND was decreased in patients (1.86 ± 0.05) to 86% of control values (2.14 ± 0.08) (P = 0.01). In controls, but not in patients, BPND correlated with stimulated insulin secretion (r2 = 0.50, P = 0.03). The average functional binding capacity was decreased by at least 40% in patients (P = 0.001). The changes in functional binding capacity and BPND were less than the near-complete loss of stimulated insulin secretion observed in patients (P = 0.001). Conclusion: These results suggest that 11C-dihydrotetrabenazine PET allows quantification of VMAT2 binding in the human pancreas. However, BPND and functional binding capacity appear to overestimate β-cell mass given the near-complete depletion of β-cell mass in long-standing type 1 diabetes, which may be due to higher nonspecific binding in the pancreas than in the renal cortex.

VMAT2 gene expression and function as it applies to imaging β-cell mass

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia. The two main forms of the disease are distinguished by different pathogenesis, natural histories, and population distributions and indicated as either type 1 (T1DM) or type 2 diabetes mellitus (T2DM). It is well established that T1DM is an autoimmune disease whereby β-cells of pancreatic islets are destroyed leading to loss of endogenous insulin production. Albeit less dramatic, β-cell mass (BCM) also drops in T2DM. Therefore, it is realistic to expect that noninvasive measures of BCM might provide useful information in the diabetes-care field. Preclinical studies have demonstrated that BCM measurements by positron emission tomography scanning, using the vesicular monoamine transporter type 2 (VMAT2) as a tissue-specific surrogate marker of insulin production and [11C] Dihydrotetrabenazine (DTBZ) as the radioligand specific for this molecule, is feasible in animal models. Unfortunately, the mechanisms underlying β-cell-specific expression of VMAT2 are still largely unexplored, and a much better understanding of the regulation of VMAT2 gene expression and of its function in β-cells will be required before the full utility of this technique in the prediction and treatment of individuals with diabetes can be understood. In this review, we summarize much of what is understood about the regulation of VMAT2 and identify questions whose answers may help in understanding what measurements of VMAT2 density mean in the context of diabetes.

Transcript profiling of human dendritic cells maturation‐induced under defined culture conditions: comparison of the effects of tumour necrosis factor alpha, soluble CD40 ligand trimer and interferon gamma

Using cDNA arrays, we characterized patterns of gene expression in populations of human dendritic cells (DCs) produced for clinical use. Culture and maturation induction of myeloid adherent cells under serum‐free conditions yielded DCs with phenotypes similar to those described in serum‐based systems. Analysis of gene expression in DCs treated with tumour necrosis factor alpha, soluble CD40L trimer or interferon gamma, however, showed specific patterns for each factor examined. Our studies document the expression of several transcripts that have not hitherto been described in DCs and/or differentially regulated according to the differentiation state of the DCs, and suggest important functional differences among the DC populations examined. In addition, DC maturation directs changes in the levels of mRNA specific for transcriptional regulators that effect the production of cytokines (e.g. BCL‐6, c‐rel). Other changes observed, including alteration in the gene expression profile of adhesion molecules and chemokine receptors such as CD44H, CD 49B, Rantes R, CXCR5 and CD37, suggest differences in trafficking potential between the populations studied. This broad‐based description of DC populations, produced under serum‐free conditions, has enabled us to better define intermediate stages of DC maturation as well as the differentiation‐inducing effects of cytokines on these cells.

Islet grafting and imaging in a bioengineered intramuscular space

Background. Because the hepatic portal system may not be the optimal site for islet transplantation, several extrahepatic sites have been studied. Here, we examine an intramuscular transplantation site, bioengineered to better support islet neovascularization, engraftment, and survival, and we demonstrate that at this novel site, grafted beta cell mass may be quantitated in a real-time noninvasive manner by positron emission tomography (PET) imaging. Methods. Streptozotocin-induced rats were pretreated intramuscularly with a biocompatible angiogenic scaffold received syngeneic islet transplants 2 weeks later. The recipients were monitored serially by blood glucose and glucose tolerance measurements and by PET imaging of the transplant site with [11C] dihydrotetrabenazine. Parallel histopathologic evaluation of the grafts was performed using insulin staining and evaluation of microvasularity. Results. Reversal of hyperglycemia by islet transplantation was most successful in recipients pretreated with bioscaffolds containing angiogenic factors when compared with those who received no bioscaffolds or bioscaffolds not treated with angiogenic factors. PET imaging with [11C] dihydrotetrabenazine, insulin staining, and microvascular density patterns were consistent with islet survival, increased levels of angiogenesis, and with reversal of hyperglycemia. Conclusions. Induction of increased neovascularization at an intramuscular site significantly improves islet transplant engraftment and survival compared with controls. The use of a nonhepatic transplant site may avoid intrahepatic complications and permit the use of PET imaging to measure and follow transplanted beta cell mass in real time. These findings have important implications for effective islet implantation outside of the liver and offer promising possibilities for improving islet survival, monitoring, and even prevention of islet loss.

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.

Suppression of the indirect pathway of T cell reactivity by high doses of allopeptide

T helper cells, which recognize allopeptides processed and presented by self APC, contribute to the generation of both cellular and humoral immune responses against allogeneic transplants. We have explored the hypothesis that the indirect T cell recognition pathway is initiated by soluble MHC antigens and that it can be suppressed by high doses of synthetic peptides corresponding to the dominant alloepitope. T cells from a DR11/7 responder were immunized in vitro with recombinant HLA-DR4 (rDR4). Experiments using partially overlapping synthetic peptides showed that the resulting T cell line (TCL) recognized a single dominant epitope mapping within residues 69-88 of the first domain of the DR4 molecule. In vitro immunization with synthetic allopeptides corresponding to other polymorphic regions, were unable to elicit T cell reactivity against rDR4, although at least one of these peptides (corresponding to residues 13-27) was immunogenic, behaving like a cryptic epitope. The rDR4-specific TCL expressed a limited TCR repertoire and provided help to autologous B cells for the production of specific antibodies. The T cell blastogenic response as well as the transcription and secretion of IL-4 (but not IL-2) was efficiently suppressed by high doses of the dominant allopeptide. These findings support the concept that selective immunointervention of indirect allorecognition can be achieved by use of high doses of antigen or TCR vaccination, as proposed for autoimmune diseases.

Tissue-Specific Self-Peptides Bound by Major Histocompatibility Complex Class I Molecules of a Human Pancreatic β-cell Line

The process of β-cell destruction in IDDM is mediated, in part, by CD8+ T-cells. Structural characterization of HLA-I-bound self-peptides presented by the human β-cell line HP-62 was performed to identify possible tissue-specific autoantigens in the context of CD8+ T-cell/HLA-I interactions. The sequences of the β-cell line HLA-I-bound peptides were compared with sequence databases. Six of the obtained sequences showed homology to known precursor proteins, three of which—GLUT2 receptor, phosphatidylinositol-glycan-specific phospholipase D, and 5-hydroxytryptamine-1F receptor—have a limited, tissue-specific expression. These HLA-bound self-peptides may be part of a pool of autoantigens recognized by β-cell reactive cytotoxic T-cells.

Contrasting effects of IFNα on MHC class II expression in professional vs. nonprofessional APCs: Role of CIITA type IV promoter

We previously demonstrated that, in ex vivo cultures, IFNα downregulates the expression of MHC class II (MHCII) genes in human non-professional APCs associated with pancreatic islets. IFNα has an opposing effect on MHCII expression in professional APCs. In this study, we found that the mechanism responsible for the IFNα-mediated MHCIIs downregulation in human MHCII-positive non-professional antigen presenting human non-hematopoietic cell lines is the result of the negative feedback system that regulates cytokine signal transduction, which eventually inhibits promoters III and IV of CIITA gene. Because the CIITA-PIV isoform is mostly responsible for the constitutive expression of MHCII genes in non-professional APCs, we pursued and achieved the specific knockdown of CIITA-PIV mRNA in our in vitro system, obtaining a partial silencing of MHCII molecules similar to that obtained by IFNα. We believe that our results offer a new understanding of the potential significance of CIITA-PIV as a therapeutic target for interventional strategies that can manage autoimmune disease and allograft rejection with little interference on the function of professional APCs of the immune system.

Anti-incretin, Anti-proliferative Action of Dopamine on β-Cells.

Human islet β-cells exploit an autocrine dopamine (DA)-mediated inhibitory circuit to regulate insulin secretion. β-Cells also express the DA active transporter and the large neutral amino acid transporter heterodimer enabling them to import circulating DA or its biosynthetic precursor, L-3,4-dihydroxyphenylalanine (L-DOPA). The capacity to import DA or L-DOPA from the extracellular space possibly indicates that DA may be an endocrine signal as well. In humans, a mixed meal stimulus is accompanied by contemporary serum excursions of incretins, DA and L-DOPA, suggesting that DA may act as an anti-incretin as postulated by the foregut hypothesis proposed to explain the early effects of bariatric surgery on type 2 diabetes. In this report, we take a translational step backwards and characterize the kinetics of plasma DA and incretin production after a mixed meal challenge in a rat model and study the integration of incretin and DA signaling at the biochemical level in a rodent β-cell line and islets. We found that there are similar excursions of incretins and DA in rats, as those reported in humans, after a mixed meal challenge and that DA counters incretin enhanced glucose-stimulated insulin secretion and intracellular signaling at multiple points from dampening calcium fluxes to inhibiting proliferation as well as apoptosis. Our data suggest that DA is an important regulator of insulin secretion and may represent 1 axis of a gut level circuit of glucose and β-cell mass homeostasis.