Need for Better Diabetes Treatment: The Therapeutic Potential of NMDA Receptor Antagonists.

Diabetes mellitus is the most common metabolic disorder in children and adolescents. Optimal control of blood glucose concentration is essential to prevent acute and diabetic long-term complications. The options to treat diabetes have clearly improved over the last decades, however, to date neither type 1 diabetes nor type 2 diabetes mellitus can be cured. Therefore, diabetes research aims at developing ß-cell protective agents that prevent or even reverse diabetes onset. N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are widely expressed in the central nervous system (CNS) where they hold central roles in CNS function. NMDAR dysfunction is associated with several neurological and psychiatric disorders and therefore NMDAR modulators have several potential therapeutic indications. Only little is known about the role of pancreatic NMDA receptors. Our data provide evidence that inhibition of pancreatic NMDARs, either genetically or pharmacologically with the over-the-counter drug dextromethorphan, increases glucose-stimulated insulin secretion from mouse and human pancreatic islets, improves glucose tolerance in mice and individuals with diabetes and promotes islet cell survival under diabetogenic conditions. Thus, our data indicate for the first time that NMDAR antagonists could serve as adjunct treatment for diabetes mellitus. The development of a safe, blood glucose lowering and particularly ß-cell protective medication would significantly enhance current diabetes treatment.

Effects of dextromethorphan as add-on to sitagliptin on blood glucose and serum insulin concentrations in individuals with type 2 diabetes mellitus: a randomized, placebo-controlled, double-blinded, multiple crossover, single-dose clinical trial.

In this clinical trial, we investigated the blood glucose (BG)-lowering effects of 30, 60 and 90 mg dextromethorphan (DXM) as well as 100 mg sitagliptin alone versus combinations of DXM and sitagliptin during an oral glucose tolerance test (OGTT) in 20 men with T2DM. The combination of 60 mg DXM plus 100 mg sitagliptin was observed to have the strongest effect in the OGTT. It lowered maximum BG concentrations and increased the baseline-adjusted area under the curve for serum insulin concentrations in the first 30 min of the OGTT (mean ± standard deviation 240 ± 47 mg/dl and 8.1 ± 6.1 mU/l/h, respectively) to a significantly larger extent than did 100 mg sitagliptin alone (254 ± 50 mg/dl and 5.8 ± 2.5 mU/l/h, respectively; p < 0.05) and placebo (272 ± 49 mg/dl and 3.9 ± 3.0 mU/l/h, respectively; p < 0.001). All study drugs were well tolerated, alone and in combination, without serious adverse events or hypoglycaemia. Long-term clinical trials are now warranted to investigate the potential of the combination of 30 or 60 mg DXM and dipeptidyl peptidase-4 inhibitors in the treatment of individuals with T2DM, in particular as preclinical studies have identified the ß-cell protective properties of DXM.

Pharmacological inhibition of Eph receptors enhances glucose-stimulated insulin secretion from mouse and human pancreatic islets.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by impaired glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Since erythropoietin-producing hepatoma (Eph)-ephrin bidirectional signalling fine-tunes GSIS from pancreatic beta cells, we investigated Eph receptor tyrosine kinases (RTK) as potential drug targets for selectively increasing GSIS. METHODS: Insulin secretion assays were carried out using mouse and human pancreatic islets as well as mouse insulinoma (MIN6) cells in the presence or absence of two Eph RTK inhibitors. Furthermore, the most potent inhibitor was injected into mice to evaluate its effects on glucose tolerance and plasma insulin levels. RESULTS: We showed that the Eph RTK inhibitors selectively increased GSIS from MIN6 cells as well as mouse and human islets. Our results also showed that the insulin secretory effects of these compounds required Eph-ephrin signalling. Finally, pharmacological inhibition of Eph receptor signalling improved glucose tolerance in mice. CONCLUSIONS/INTERPRETATION: We showed for the first time that Eph RTKs represent targets for small molecules to selectively increase GSIS and improve glucose tolerance.

Association of exercise-induced hyperinsulinaemic hypoglycaemia with MCT1-expressing insulinoma.

AIMS/HYPOTHESIS: Exercise-induced hyperinsulinism (EIHI) is a hypoglycaemic disorder characterised by inappropriate insulin secretion following anaerobic exercise or pyruvate load. Activating promoter mutations in the MCT1 gene (also known as SCLA16A1), coding for monocarboxylate transporter 1 (MCT1), were shown to associate with EIHI. Recently, transgenic Mct1 expression in pancreatic beta cells was shown to introduce EIHI symptoms in mice. To date, MCT1 has not been demonstrated in insulin-producing cells from an EIHI patient. METHODS: In vivo insulin secretion was studied during an exercise test before and after the resection of an insulinoma. The presence of MCT1 was analysed using immunohistochemistry followed by laser scanning microscopy, western blot analysis and real-time RT-PCR of MCT1. The presence of MCT1 protein was analysed in four additional insulinoma patients. RESULTS: Clinical testing revealed massive insulin secretion induced by anaerobic exercise preoperatively, but not postoperatively. MCT1 protein was not detected in the patient's normal islets. In contrast, immunoreactivity was clearly observed in the insulinoma tissue. Western blot analysis and real-time RT-PCR showed a four- to fivefold increase in MCT1 in the insulinoma tissue of the EIHI patient compared with human pancreatic islets. MCT1 protein was detected in three of four additional insulinomas. CONCLUSIONS/INTERPRETATION: We show for the first time that an MCT1-expressing insulinoma was associated with EIHI and that MCT1 might be present in most insulinomas. Our data suggest that MCT1 expression in human insulin-producing cells can lead to EIHI and warrant further studies on the role of MCT1 in human insulinoma patients.

Cell-cell interactions in the endocrine pancreas.

Cell-cell communication within any given tissue is an important aspect of correct organ function. The islets of Langerhans forming the endocrine pancreas are composed of alpha-, beta-, delta-, epsilon- and PP-cells, and interactions between these cells are required for fine-tuning glucose homeostasis of the body. The endocrine cells communicate through homotypic or heterotypic cell-cell adhesion, or in a paracrine fashion, and this communication is involved in the regulated secretion of islet hormones. This review discusses how islet hormones, secreted molecules and ions influence the endocrine cells and how cell adhesion molecules such as neural cell adhesion molecule, cadherins, connexin-36, Eph receptors and ephrin ligands, as well as extracellular matrix proteins, modulate pancreatic islet function.

The vascular trigger of type II diabetes mellitus.

Type II diabetes mellitus is associated with obesity and insulin resistance. However, many humans with these symptoms never develop diabetes. This raises the key question of the difference that determines why one obese individual develops diabetes, whereas another one does not. Here we review the experimental support for our hypothesis that mutual signaling between insulin producing beta cells and pancreatic endothelial cells determines whether a person develops type II diabetes. According to our hypothesis, a disturbance of this mutual signaling leads to diabetes in an insulin-resistant individual. We discuss that most type II diabetes-associated genes have a function in the vascular system, and that endoplasmic reticulum (ER) stress in beta cells can result from vascular defects in pancreatic islets. Whereas vascular complications have been widely accepted as a result of type II diabetes, we suggest that changes within the vascular system may act at an earlier stage and trigger the onset of type II diabetes.

Reduced insulin secretion and content in VEGF-a deficient mouse pancreatic islets.

Mice, deficient for vascular endothelial growth factor VEGF-A in pancreatic islets, have reduced insulin gene expression levels and an impaired glucose tolerance. Here, we investigated whether VEGF-A was required for physiological glucose-stimulated insulin secretion and insulin content. We performed in situ pancreas perfusions and islet perifusions on mice lacking VEGF-A in the pancreatic epithelium in order to study their ability to secrete insulin in response to glucose. We identified insulin secretion defects in the pancreata of VEGF-A deficient mice, including a delayed and blunted response to glucose. Islet perifusion experiments revealed a missing first phase and weaker second phase of insulin secretion, in two of three VEGF-A deficient mice. On average, insulin content in VEGF-A deficient islets was significantly reduced when compared with control islets. We conclude that VEGF-A is required in pancreatic islets for normal glucose-stimulated insulin secretion and physiological insulin content. Thus, VEGF-A is a key factor for pancreatic islet function.

Induction of pancreatic differentiation by signals from blood vessels.

Blood vessels supply developing organs with metabolic sustenance. Here, we demonstrate a role for blood vessels as a source of developmental signals during pancreatic organogenesis. In vitro experiments with embryonic mouse tissues demonstrate that blood vessel endothelium induces insulin expression in isolated endoderm. Removal of the dorsal aorta in Xenopus laevis embryos results in the failure of insulin expression in vivo. Furthermore, using transgenic mice, we show that ectopic vascularization in the posterior foregut leads to ectopic insulin expression and islet hyperplasia. These results indicate that vessels not only provide metabolic sustenance, but also provide inductive signals for organ development.

Notch gene expression during pancreatic organogenesis.

Notch receptors are involved in regulating the balance between cell differentiation and stem cell proliferation during the development of numerous tissues (Artavanis-Tsakonas, S., Matsuno, K., Fortini, M. E., 1995. Notch signaling. Science 268, 225-232). Here the expression of all four vertebrate Notch genes, their ligands, and some down-stream targets is analyzed during mouse pancreatic organogenesis. Notch 1 is the first Notch gene expressed in the pancreatic epithelium, and coexpression with HES 1 suggests that the Notch 1 pathway is activated. Notch 2 expression follows later when pancreatic buds branch and is restricted to embryonic ducts, believed to be the source for endocrine and exocrine stem cells. Notch 3 and Notch 4 are expressed in pancreatic mesenchyme and later in endothelial cells. Together these descriptive data comprise a framework for understanding the cellular basis for Notch function during pancreatic development.

The role of peptide presentation in the physiological function of HLA-G.

The HLA-G gene gives rise to six differently spliced mRNAs. The membrane bound HLA-G1 molecule containing all three extracellular domains presents peptides that follow motif requirements similar to those of classical HLA class I molecules. This isoform is also capable of inhibiting Natural Killer (NK) cells, but is only efficiently transported to the cell surface when peptides are provided in the endoplasmic reticulum. In the absence of sufficient peptide supply to the ER a small molecule of 18-kDa is transported to the cell surface in HLA-G transfectants of LCL721.221 cells. HLA-G transfectants with impaired ER peptide supply are nevertheless protected from NK lysis.

Stress proteins and immunity mediated by cytotoxic T lymphocytes.

Chaperone molecules, including members of the heat shock protein family, are able to stimulate alphabeta and gammadelta T cells as well as natural killer cells. For alphabeta T cells, specificity is induced by chaperone-assisted peptides; this has lead to detailed investigations of peptides that bind to these chaperones and their possible role in antigen presentation.

Cell lines transfected with the TAP inhibitor ICP47 allow testing peptide binding to a variety of HLA class I molecules.

The immediate early protein ICP47 of the Herpes simplex virus is known to block the human transporter associated with antigen processing (TAP), thereby creating a TAP-deficient phenotype in any human cell transfected with the corresponding cDNA. Exploiting this inhibitory activity, we constructed a selection of human cell lines each co-expressing one of the cDNAs of human leukocyte antigen (HLA) class I alleles HLA-A*1101, A24, A*3101, A*6601, B8 and B*1516, and the cDNA encoding the ICP47 molecule. The cell lines generated showed diminished HLA class I surface expression and the inhibition of the TAP function was confirmed in peptide translocation assays. The addition of specific exogenous peptide ligands restored the expression of the corresponding HLA class I molecules. Thus, the ICP47 transfectants provide us with a tool to closely examine peptide-HLA class I interactions, to confirm HLA class I ligand motifs and to test peptides predicted to bind.

Protein disulfide isomerase is the dominant acceptor for peptides translocated into the endoplasmic reticulum.

Peptides derived from cytosolic protein degradation are translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). In the ER, class I molecules bind the peptides fitting to their respective motifs and present them on the cell surface to CD8+ T lymphocytes. However, most TAP-translocated peptides are not expected to bind to the class I molecules present in a particular cell. Recently, we have demonstrated that TAP-translocated peptides containing a photoreactive phenylalanine analogue can be cross-linked to two luminal ER-resident proteins: with low efficiency to the stress protein gp96 and with high efficiency to a 60-kDa protein (Lammert, E. et al., Eur. J. Immunol. 1997. 27: 923). Both proteins have also been labeled specifically by TAP-translocated peptides conjugated to a different photoreactive group (Marusina, K. et al., Biochemistry 1997. 36: 856). Here, we show that the 60-kDa peptide-binding protein is identical to the multifunctional protein disulfide isomerase (PDI). Since PDI is the only luminal ER-resident protein that is labeled by the photoreactive peptides with high efficiency, it might represent the dominant acceptor for TAP-translocated peptides.

The endoplasmic reticulum-resident stress protein gp96 binds peptides translocated by TAP.

The endoplasmic reticulum (ER)-resident stress protein gp96 induces a major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response against antigens present in the cells from which it has been prepared. In this study, photoreactive peptides were translocated into the ER by the transporter associated with antigen processing (TAP). These peptides can be cross-linked specifically to gp96. Thus, we provide the first evidence that gp96 binds TAP-translocated peptides which have been implicated in the induction of specific CTL responses after immunization with gp96 (Srivastava, P. K. et al., Immunogenetics 1994. 39: 93).

Expression levels of stress protein gp96 are not limiting for major histocompatibility complex class I-restricted antigen presentation.

Immunization of mice with gp96 induces CTL with specificity for proteins that are expressed in the cells from which gp96 was isolated (Arnold et al., J. Exp. Med. 1995. 182: 885, Udono et al., Proc. Natl. Acad. Sci. USA 1994. 91: 3077). Recently, it has been shown that gp96 from cells transfected with vesicular stomatitis virus (VSV) nucleocapsid protein as well as gp96 loaded in vitro with peptides containing an epitope of this protein are taken up by phagocytic cells which obtain thereby the capacity for stimulating VSV-specific cytotoxic T lymphocytes (Suto and Srivastava, Science 1995. 269: 1585). The immunization experiments together with the peptide transfer from gp96/peptide complexes to major histocompatibility complex (MHC) class I molecules of phagocytic cells are consistent with the hypothesis that the endoplasmic reticulum-resident protein gp96 plays a crucial role in the antigen presentation of a cell (Srivastava et al., Immunogenetics 1994. 29: 93). To examine the involvement of gp96 in class I-restricted antigen presentation, we reduced gp96 RNA and protein levels by transfecting P13.1 cells with a vector containing part of gp96 cDNA in antisense orientation to the promotor. We found that antisense clones expressing strongly reduced levels of gp96 mRNA and gp96 protein show normal levels of MHC class I molecules on the cell surface and are recognized by T cells to the same extent as wild-type cells. Thus, our results show that normal levels of gp96 expression in a cell are not limiting for class I-restricted antigen presentation.