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Although, diabetes is reaching pandemic proportions, the exact aetiology of either type 1 ( T1D) or type 2 diabetes ( T2D) remains to be determined. Mounting evidence, however, suggests that islet inflammation is a likely common denominator during early development of either type of the disease. In this review, we highlight some of the inflammatory mechanisms that appear to be shared between T1D and T2D, and we explore the utility of intravital imaging in the study of islet inflammation. Intravital imaging has emerged as an indispensable tool in biomedical research and a variety of in vivo imaging approaches have been developed to study pancreatic islet physiology and pathophysiology in the native environment in health and disease. However, given the scattered distribution of the islets of Langerhans within the 'sea' of the exocrine pancreas located deep within the body and the fact that the islets only constitute 1-2% of the total volume of pancreatic tissue, studying the pancreatic islet in situ has been challenging. Here, we focus on a new experimental approach that enables studying local islet inflammation with single-cell resolution in the relevant context of the in vivo environment non-invasively and longitudinally and, thereby improving our understanding of diabetes pathogenesis. [ABSTRACT FROM AUTHOR]

A major problem in medical research is to translate in vitro observations into the living organism. In this perspective, we discuss ongoing efforts to non-invasively image pancreatic islets/β-cells by techniques, such as magnetic resonance imaging and positron emission tomography, and present an experimental platform, which allows in vivo imaging of pancreatic β-cell mass and function longitudinally and at the single-cell level. Following transplantation of pancreatic islets into the anterior chamber of the eye of mice and rats, these islets are studied by functional microscopic imaging. This imaging platform can be utilized to address fundamental aspects of pancreatic islet cell biology in vivo in health and disease. These include the dynamics of pancreatic islet vascularization, islet cell innervation, signal-transduction, change in functional β-cell mass and immune responses. Moreover, we discuss the feasibility of studying human islet cell physiology and pathology in vivo as well as the potential of using the anterior chamber of the eye as a site for therapeutic transplantation in type 1 diabetes mellitus. [ABSTRACT FROM AUTHOR]

An insufficient number of insulin-producing β-cells is a major cause of defective control of blood glucose in both type 1 and type 2 diabetes. The aim of this study was to clarify whether the insulinotropic imidazolines can affect the survival of highly proliferating insulin-secreting cells, here exemplified by the MIN6 cell line. Our data demonstrate that RX871024, but not efaroxan, triggered MIN6 cell death and potentiated death induced by a combination of the pro-inflammatory cytokines interleukin-1β, interferon- γ and tumor necrosis factor-α. These effects did not involve changes in nitric oxide production but correlated with stimulation of c-jun N-terminal kinase (JNK) activity and activation of caspases-1, -3, -8 and -9. Our results suggest that the imidazoline RX871024 causes death of highly proliferating insulin-secreting cells, putatively via augmentation of JNK activity, a finding that may impact on the possibility of using compounds of similar activity in the treatment of diabetes. [ABSTRACT FROM AUTHOR]

The mechanism by which the novel, pure glucose-dependent insulinotropic, imidazoline derivative BL11282 promotes insulin secretion in pancreatic islets has been investigated. The roles of KATP channels, α2-adrenoreceptors, the I1-receptor-phosphatidylcholine-specific phospholipase (PC-PLC) pathway and arachidonic acid signaling in BL11282 potentiation of insulin secretion in pancreatic islets were studied. Using SUR1(-/-) deficient mice, the previous notion that the insulinotropic activity of BL11282 is not related to its interaction with KATP channels was confirmed. Insulinotropic activity of BL11282 was not related to its effect on α2-adrenoreceptors, I1-imidazoline receptors or PC-PLC. BL11282 significantly increased [3H]arachidonic acid production. This effect was abolished in the presence of the iPLA2 inhibitor, bromoenol lactone. The data suggest that potentiation of glucose-induced insulin release by BL11282, which is independent of concomitant changes in cytoplasmic free Ca2+ concentration, involves release of arachidonic acid by iPLA2 and its metabolism to epoxyeicosatrienoic acids through the cytochrome P-450 pathway. [ABSTRACT FROM AUTHOR]

The effects of an imidazoline compound (BL11282) on protein expression in rat pancreatic islets were investigated with a proteomic approach. The compound increases insulin release selectively at high glucose concentrations and is therefore of interest in type 2 diabetes. Whole cell extracts from isolated drug-treated and native pancreatic rat islets were compared after separation by 2-D gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry; 15 proteins were selectively up-regulated and 7 selectively down-regulated in drug-treated islets. Of special interest among the differentially expressed proteins are those involved in protein folding (Hsp60, protein disulfide isomerase, calreticulin), Ca2+ binding (calgizzarin, calcyclin and annexin I) and metabolism or signalling (pyruvate kinase, alpha enolase and protein kinase C inhibitor 1). [ABSTRACT FROM AUTHOR]

The recently reported influence of proinsulin C-peptide on insulin prompted us to examine structural features of the C-peptide. Four sets of limited pattern similarities towards inter-chain end regions of insulin were noticed, involving secondary structure elements, binding residues and intra- as well as inter-peptide residue similarities. Using surface plasmon resonance, we examined insulin binding to truncated, soluble insulin receptor A and IGF-1 receptor, but C-peptide effects on these bindings were not detectable. Two forms of the insulin receptor, differing in activation of gene transcription with regards to (pre)proinsulin and glucokinase, respectively, were also uninfluenced by C-peptide. We conclude that the pattern similarities, if functional, reflect C-peptide interactions with molecules other than both insulin A and B receptors and IGF-1 receptors. Any such effects are of interest in relation to reported binding interactions between insulin and C-peptide. [ABSTRACT FROM AUTHOR]

The insulinotropic activity of the novel imidazoline compound BL11282 was investigated. Intravenous administration of BL11282 (0.3 mg x kg(-1) x min(-1)) to anesthetized rats did not change blood glucose and insulin levels under basal conditions, but produced a higher increase in blood insulin levels and a faster glucose removal from the blood after glucose infusion. Similarly, in isolated Wistar rat pancreatic islets, 0.1-100 micromol/l BL11282 potently stimulated glucose-induced insulin secretion but did not modulate basal insulin secretion. Unlike previously described imidazolines, BL11282 did not block ATP-dependent K+ channels. Furthermore, the compound stimulated insulin secretion in islets depolarized with high concentrations of KCl or permeabilized with electric shock. Insulinotropic activity of BL11282 was dependent on activity of protein kinases A and C. In pancreatic islets from spontaneously diabetic GK rats, the imidazoline compound restored the impaired insulin response to glucose. In conclusion, the imidazoline BL11282 constitutes a new class of insulinotropic compounds that exerts an exclusive glucose-dependent insulinotropic activity in pancreatic islets by stimulating insulin exocytosis. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is an autoimmune disease of unknown etiology. Our previous work has shown that a factor present in serum from type 1 diabetic patients causes increased Ca2+ channel activity and apoptotic DNA fragmentation in pancreatic beta-cells. Here we examined the effects of type 1 diabetic serum on primary cerebellar granule cells (CGCs). In CGCs, exposure to type 1 diabetic serum did not cause increased apoptosis or changes in Ca2+ channel activity. However, patient serum did cause modulation of Ca2+ signals in a cell type with triangular soma that exhibited low voltage-gated Ca2+ currents. This cell was present primarily in cultures exposed to type 1 diabetic serum. The presence of low voltage-gated Ca2+ currents and long neuronal dendrites indicated that this unique cell was of neuronal origin and not of glial origin. [ABSTRACT FROM AUTHOR]

Imidazoline compounds have been considered for the treatment of type 2 diabetes. We have now investigated the effects of imidazolines on interleukin (IL)-1beta-induced beta-cell apoptosis and the signal transduction pathways involved. Inhibition of Ca2+ influx into beta-cells by D-600, a blocker of voltage-gated L-type Ca2+ channels, suppressed IL-1beta-induced apoptosis. Our data show that calcineurin, Ca2+/calmodulin-dependent serine/threonine protein phosphatase 2B, is responsible for the effect of Ca2+ on beta-cell apoptosis. We also demonstrate that IL-1beta-mediated apoptosis correlates with expression of inducible nitric oxide synthase (iNOS) and the increase in intracellular production of nitric oxide. An inhibitor of cGMP-dependent protein kinase (PKG), KT5823, suppressed IL-1beta-induced apoptosis, suggesting the involvement of a PKG-dependent pathway in the apoptotic process. One of the major findings in this study is that imidazoline compounds RX871024 and efaroxan, suggested as prototypes of a new generation of drugs against type 2 diabetes, can protect against IL-1beta-induced apoptosis in pancreatic beta-cells, possibly by their inhibition of the expression of iNOS, a key element in the IL-1beta-induced apoptotic pathway in pancreatic beta-cells. These data suggest that imidazoline compounds should be explored as a potential therapeutic agent for the treatment of both type 1 and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Apoptosis is a physiological form of cell death that occurs during normal development, and critical mediators of this process include caspases, reactive oxygen species, and Ca2+. Excessive apoptosis of the pancreatic beta-cell has been associated with diabetes. Consequently, apoptosis research has focused on how infiltrating macrophages or cytotoxic T-cells might kill pancreatic beta-cells using cytokines or death receptor triggering. Meanwhile, the intracellular events in the target beta-cell have been largely ignored. Elucidation of such targets might help develop improved treatment strategies for diabetes. This article will outline recent developments in apoptosis research, with emphasis on mechanisms that may be relevant to beta-cell death in type 1 and type 2 diabetes. Several of the models proposed in beta-cell killing converge on Ca2+ signaling, indicating that the pancreatic beta-cell may be an ideal system in which to carefully dissect the role of Ca2+ during apoptosis. [ABSTRACT FROM AUTHOR]

Fluorescent proteins have been extensively used as protein "tags" to study the subcellular localization of proteins and/or their translocation upon stimulation or as markers for transfection in transient and stable expression systems. However, they have not been frequently used as reporter genes to monitor stimulus-induced gene expression in mammalian cells. Here we demonstrate the use of fluorescent proteins to study stimulus-induced gene transcription. The general applicability of the approach is exemplified by doxycyclin-(Tet-On) and phorbol 12-myristate 13-acetate-induced (c-fos) promoter activation, with green fluorescent protein (GFP) and red fluorescent protein (DsRed) as semiquantitative and immediate reporters, of transcription activation. Under the control of beta-cell-specific promoters, such as the rat insulin 1 promoter or the rat upstream glucokinase promoter, this approach allowed us to monitor online glucose-induced gene transcription in primary beta-cells at the single-cell level as well as in the context of the islet of Langerhans. Applying discretely detectable fluorescent proteins, for example GFP and DsRed, enabled us to simultaneously monitor stimulus-induced transcription by two different promoters in the same cell. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been suggested to play an important role as an endogenous regulator of ATP-sensitive potassium (KATP) channels consisting of Kir6.2 as a pore-forming subunit. These studies show the ability of PIP2 to activate KATP channel activity and to counteract the inhibitory effect of ATP, implying that PIP2 could serve the function of modulating the sensitivity of KATP channels to the cytoplasmic free ATP concentration. Careful examination of the literature reveals that the definitive physiologically relevant experiments to establish efficacy of PIP2 on this channel may still have to be performed. Our reservations are based on the handling of PIP2 in cell-free experiments and in various strategies designed to modulate PIP2 concentrations in intact cells. Furthermore, a potent stimulatory effect of phosphatidylinositol 3,4,5trisphosphate, a downstream metabolite of PIP2, on KATP channel activity raises the possibility that the effects on the KATP channel may not be directly related to PIP2. [ABSTRACT FROM AUTHOR]

Synaptotagmin is involved in Ca2+-regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic beta-cell is an example of a Ca2+-dependent secretory process. Previous studies of pancreatic beta-cells were unable to show presence of synaptotagmin I. We now present biochemical and immunohistochemical data showing that synaptotagmin III is present in pancreatic beta-cells as well as in the insulin-secreting cell line HIT-T15 and in rat insulinoma. By subcellular fractionation, we found synaptotagmin III in high-density fractions together with insulin and secretogranin I, indicating colocalization of synaptotagmin III and insulin in secretory granules. We could also show that blockade of synaptotagmin III by a specific antibody inhibited Ca2+-induced changes in beta-cell membrane capacitance, suggesting that synaptotagmin III is part of the functional protein complex regulating beta-cell exocytosis. The synaptotagmin III antibody did not affect the activity of the voltage-gated L-type Ca2+-channel. These findings are compatible with the view that synaptotagmin III, because of its distinct localization in the pancreatic beta-cell, functionally modulates insulin exocytosis. This indicates that synaptotagmin may have a general role in the regulation of exocytosis not only in neuronal cells but also in endocrine cells. [ABSTRACT FROM AUTHOR]

The T-type Ca2+ channel is an important determinant of electrical activity and of Ca2+ influx in rat and human pancreatic beta-cells. We have identified and sequenced a cDNA encoding a T-type Ca2+ channel alpha1-subunit derived from INS-1, the rat insulin-secreting cell line. The sequence of the cDNA indicates a protein composed of 2,288 amino acids that shares 96.3% identity to alpha1G, the neuronal T-type Ca2+ channel subunit. The transmembrane domains of the protein are highly conserved, but the isoform contains three distinct regions and 10 single amino acid substitutions in other regions. Sequencing rat genomic DNA revealed that the alpha1-subunit we cloned is an alternative splice isoform of alpha1G. By using specific primers and reverse transcription-polymerase chain reaction, we demonstrated that both splice variants are expressed in rat islets. The isoform deduced from INS-1 was also expressed in brain, neonatal heart, and kidney. Functional expression of this alpha1G isoform in Xenopus oocytes generated low voltage-activated Ba2+ currents. These results provide the molecular biological basis for studies of function of T-type Ca2+ channels in beta-cells, which is where these channels may play critical roles in diabetes. [ABSTRACT FROM AUTHOR]

The effects of RX871024, a compound with an imidazoline structure, on cytoplasmic-free Ca2+ concentration ([Ca2+]i) in mouse pancreatic beta-cells were studied. RX871024 modulates [Ca2+]i by at least two mechanisms. One mechanism involves closure of ATP-regulated K+ channels, resulting in membrane depolarization, opening of voltage-gated L-type Ca2+ channels, and a subsequent increase in [Ca2+]i. Another mechanism, reported here for the first time, deals with RX871024-induced mobilization of Ca2+ from nonmitochondrial thapsigargin-sensitive intracellular stores. Reduced glutathione, inhibitors of cytochrome P-450, and monoaminooxidases A and B blocked this Ca2+ mobilization. It is concluded that the mechanism of RX871024-induced Ca2+ mobilization from intracellular stores involves changes in the oxidation/reduction state of the pancreatic beta-cell and may be controlled by cytochrome P-450. [ABSTRACT FROM AUTHOR]

Recently, it has been proposed that leptin, the ob gene product, influences some steps in the insulin-signaling cascade. The purpose of the present study was to determine whether leptin exerts direct effects on glucose transport in insulin target tissues. Epitrochlearis muscles or isolated adipocytes from male SD rats were incubated in the absence or presence of recombinant leptin (3-1,000 ng/ml), and in the absence or presence of submaximal or maximal insulin concentrations. In skeletal muscle, insulin increased 3-O-methylglucose transport (1.88 +/- 0.21, 4.06 +/- 0.59, and 9.35 +/- 1.90 micromol x ml-1 x h-1, for 0, 0.6, and 12.0 nmol/l insulin, respectively). Leptin exposure (300 ng/ml) for 2 h did not alter the basal, submaximal, or maximal response of glucose transport to insulin in skeletal muscle (1.50 +/- 0.14, 4.76 +/- 0.58, and 9.04 +/- 1.09 micromol x ml-1 x h-1 for 0, 0.6, and 12.0 nmol/l insulin, respectively). Insulin increased glucose transport in rat adipocytes (0.194 +/- 0.007, 1.059 +/- 0.029, and 3.367 +/- 0.143 pmol [14C]glucose x 0.5 ml-1 cell suspension x min-1 for 0, 0.8, and 80 nmol/l insulin, respectively); in vitro exposure to leptin (300 ng/ml) did not alter glucose transport (0.220 +/- 0.006, 1.269 +/- 0.046, and 3.221 +/- 0.285 pmol [14C]glucose x 0.5 ml-1 cell suspension x min-1 for 0, 0.8, and 80 nmol/l insulin, respectively). Similar to our findings in the epitrochlearis muscle, leptin had no direct effect on basal or insulin-stimulated glucose uptake in soleus muscle from ob/ob or lean mice or adipocytes from normal mice. In summary, in vitro exposure of skeletal muscle or adipocytes to recombinant leptin did not alter glucose transport in the absence of insulin, nor did it affect the sensitivity or responsiveness of the glucose transport system to insulin. [ABSTRACT FROM AUTHOR]