Pancreatic ß-cell imaging in humans: fiction or option?

Diabetes mellitus is a growing worldwide epidemic disease, currently affecting 1 in 12 adults. Treatment of disease complications typically consumes ∼10% of healthcare budgets in developed societies. Whilst immune-mediated destruction of insulin-secreting pancreatic ß cells is responsible for Type 1 diabetes, both the loss and dysfunction of these cells underly the more prevalent Type 2 diabetes. The establishment of robust drug development programmes aimed at ß-cell restoration is still hampered by the absence of means to measure ß-cell mass prospectively in vivo, an approach which would provide new opportunities for understanding disease mechanisms and ultimately assigning personalized treatments. In the present review, we describe the progress towards this goal achieved by the Innovative Medicines Initiative in Diabetes, a collaborative public-private consortium supported by the European Commission and by dedicated resources of pharmaceutical companies. We compare several of the available imaging methods and molecular targets and provide suggestions as to the likeliest to lead to tractable approaches. Furthermore, we discuss the simultaneous development of animal models that can be used to measure subtle changes in ß-cell mass, a prerequisite for validating the clinical potential of the different imaging tracers.

Systemic bile acid sensing by G protein-coupled bile acid receptor 1 (GPBAR1) promotes PYY and GLP-1 release.

BACKGROUND AND PURPOSE: Nutrient sensing in the gut is believed to be accomplished through activation of GPCRs expressed on enteroendocrine cells. In particular, L-cells located predominantly in distal regions of the gut secrete glucagon-like peptide 1 (GLP-1) and peptide tyrosine-tyrosine (PYY) upon stimulation by nutrients and bile acids (BA). The study was designed to address the mechanism of hormone secretion in L-cells stimulated by the BA receptor G protein-coupled bile acid receptor 1 (GPBAR1). EXPERIMENTAL APPROACH: A novel, selective, orally bioavailable, and potent GPBAR1 agonist, RO5527239, was synthesized in order to investigate L-cell secretion in vitro and in vivo in mice and monkey. In analogy to BA, RO5527239 was conjugated with taurine to reduce p.o. bioavailability yet retaining its potency. Using RO5527239 and tauro-RO5527239, the acute secretion effects on L-cells were addressed via different routes of administration. KEY RESULTS: GPBAR1 signalling triggers the co-secretion of PYY and GLP-1, and leads to improved glucose tolerance. The strong correlation of plasma drug exposure and plasma PYY levels suggests activation of GPBAR1 from systemically accessible compartments. In contrast to the orally bioavailable agonist RO5527239, we show that tauro-RO5527239 triggers PYY release only when applied intravenously. Compared to mice, a slower and more sustained PYY secretion was observed in monkeys. CONCLUSION AND IMPLICATIONS: Selective GPBAR1 activation elicits a strong secretagogue effect on L-cells, which primarily requires systemic exposure. We suggest that GPBAR1 is a key player in the intestinal proximal-distal loop that mediates the early phase of nutrient-evoked L-cell secretion effects.

Taspoglutide, a novel human once-weekly GLP-1 analogue, protects pancreatic ß-cells in vitro and preserves islet structure and function in the Zucker diabetic fatty rat in vivo.

AIM: Glucagon-like peptide-1 (GLP-1) has protective effects on pancreatic ß-cells. We evaluated the effects of a novel, long-acting human GLP-1 analogue, taspoglutide, on ß-cells in vitro and in vivo. METHODS: Proliferation of murine pancreatic ß (MIN6B1) cells and rat islets in culture was assessed by imaging of 5-ethynyl-2'-deoxyuridine-positive cells after culture with taspoglutide. Apoptosis was evaluated with the transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labelling assay in rat insulinoma (INS-1E) cells and isolated human islets exposed to cytokines (recombinant interleukin-1ß, interferon-γ, tumour necrosis factor-α) or lipotoxicity (palmitate) in the presence or absence of taspoglutide. Islet morphology and survival and glucose-stimulated insulin secretion in perfused pancreata were assessed 3-4 weeks after a single application of taspoglutide to prediabetic 6-week-old male Zucker diabetic fatty (ZDF) rats. RESULTS: Proliferation was increased in a concentration-dependent manner up to fourfold by taspoglutide in MIN6B1 cells and was significantly stimulated in isolated rat islets. Taspoglutide almost completely prevented cytokine- or lipotoxicity-induced apoptosis in INS-1E cells (control 0.5%, cytokines alone 2.2%, taspoglutide + cytokines 0.6%, p < 0.001; palmitate alone 8.1%, taspoglutide + palmitate 0.5%, p < 0.001) and reduced apoptosis in isolated human islets. Treatment of ZDF rats with taspoglutide significantly prevented ß-cell apoptosis and preserved healthy islet architecture and insulin staining intensity as shown in pancreatic islet cross sections. Basal and glucose-stimulated insulin secretion of in situ perfused ZDF rat pancreata was normalized after taspoglutide treatment. CONCLUSIONS: Taspoglutide promoted ß-cell proliferation, prevented apoptosis in vitro and exerted multiple ß-cell protective effects on islet architecture and function in vivo in ZDF rats.

Taspoglutide, a novel human once-weekly analogue of glucagon-like peptide-1, improves glucose homeostasis and body weight in the Zucker diabetic fatty rat.

AIM: Glucagon-like peptide-1 (GLP-1) receptor agonists are a novel class of pharmacotherapy for type 2 diabetes. We investigated the effects of a novel, long-acting human GLP-1 analogue, taspoglutide, in the Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. METHODS: Blood glucose and plasma levels of insulin, peptide YY (PYY), glucose-dependent insulinotropic polypeptide (GIP) and triglycerides were measured during oral glucose tolerance tests (oGTT) conducted in ZDF rats treated acutely or chronically with a single long-acting dose of taspoglutide. Pioglitazone was used as a positive control in the chronic study. Postprandial glucose, body weight, glycaemic control and insulin sensitivity were assessed over 21 days in chronically treated animals. RESULTS: Acute treatment with taspoglutide reduced glucose excursion and increased insulin response during oGTT. In chronically treated rats, glucose excursion and levels of GIP, PYY and triglycerides during oGTT on day 21 were significantly reduced. Postprandial glucose levels were significantly lower than vehicle controls by day 15. A significant reduction in body weight gain was noticed by day 8, and continued until the end of the study when body weight was approximately 7% lower in rats treated with taspoglutide compared to vehicle. Glycaemic control (increased levels of 1,5-anhydroglucitol) and insulin sensitivity (Matsuda index) were improved by taspoglutide treatment. CONCLUSIONS: Taspoglutide showed typical effects of native GLP-1, with improvement in glucose tolerance, postprandial glucose, body weight, glycaemic control and insulin sensitivity.

Analysis of phosphorylation-dependent modulation of Kv1.1 potassium channels.

The voltage-gated potassium channel Kv1.1 contains phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC). To study Kv1.1 protein expression and cellular distribution in regard to its level of phosphorylation, the effects of PKA and PKC activation on Kv1.1 were investigated in HEK 293 cells stably transfected with Kv1.1 (HEK 293/1). Without kinase activation, HEK 293/1 cells carry unphosphorylated Kv1.1 protein in the plasma membranes, whereas large amounts of phosphorylated and unphosphorylated Kv1.1 protein were located intracellularly. Activation of PKA resulted in phosphorylation of intracellular Kv1.1 protein, followed by a rapid translocation of Kv1.1 into the plasma membrane. Patch-clamp analysis revealed an increase in current amplitude upon PKA activation and demonstrated differences in the voltage dependence of current activation between unphosphorylated and phosphorylated Kv1.1 channels. In contrast to PKA, even prolonged activation of PKC did not lead to direct phosphorylation of Kv1.1, but induced Kv1.1 protein synthesis. Thus, protein kinases have direct and indirect effects on the functional expression of voltage-gated potassium channels. Our data suggest that the synergistic action of protein kinases may play an important role in the fine-tuning of Kv channel function.

Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and KChip3.1 transcription.

The activity of dopaminergic (DA) substantia nigra (SN) neurons is essential for voluntary movement control. An intrinsic pacemaker in DA SN neurons generates their tonic spontaneous activity, which triggers dopamine release. We show here, by combining multiplex and quantitative real-time single-cell RT- PCR with slice patch-clamp electrophysiology, that an A-type potassium channel mediated by Kv4.3 and KChip3 subunits has a key role in pacemaker control. The number of active A-type potassium channels is not only tightly associated with the pacemaker frequency of individual DA SN neurons, but is also highly correlated with their number of Kv4.3L (long splice variant) and KChip3.1 (long splice variant) mRNA molecules. Consequently, the variation of Kv4alpha and Kv4beta subunit transcript numbers is sufficient to explain the full spectrum of spontaneous pacemaker frequencies in identified DA SN neurons. This linear coupling between Kv4alpha as well as Kv4beta mRNA abundance, A-type channel density and pacemaker frequency suggests a surprisingly simple molecular mechanism for how DA SN neurons tune their variable firing rates by transcriptional control of ion channel genes.

NIP domain prevents N-type inactivation in voltage-gated potassium channels.

Shaker-related voltage-gated K+ (Kv) channels are assembled from ion-conducting K(v)alpha subunits, which are integral membrane proteins, and auxiliary K(v)beta subunits. This leads to the formation of highly diverse heteromultimeric Kv channels that mediate outward currents with a wide range of time courses for inactivation. Two principal inactivation mechanisms have been recognized: C-type inactivation correlated with carboxy-terminal K(v)alpha-subunit structures, and N-type inactivation conferred by 'ball' domains in the amino termini of certain K(v)alpha and K(v)beta subunits. Assembly of heteromultimers with one or more K(v)alpha- and/or K(v)beta ball domains appears to be an essential principle of the generation of A-type Kv channel diversity. Here we show that, unexpectedly, the presence of K(v)alpha- or K(v)beta-ball domains does not dominate the gating phenotype in heteromultimers containing Kv1.6alpha subunits. These heteromultimers mediate non-inactivating currents because of the dominant-negative activity of a new type of N-type inactivation-prevention (NIP) domain present in the Kv1.6 amino terminus. Mutations in the NIP domain lead to loss of function, and its transfer to another K(v)alpha subunit leads to gain of function. Our discovery of the NIP domain, which neutralizes the activity of K(v)alpha- and K(v)beta-inactivation gates, establishes a new determinant for the gating behaviour of heteromultimeric Kv channels.

Secondary structure, stability and tetramerisation of recombinant K(V)1.1 potassium channel cytoplasmic N-terminal fragment.

The recombinant N-terminal fragment (amino acids 14-162) of a tetrameric voltage-gated potassium channel (K(V)1.1) has been studied using spectroscopic techniques. Evidence is presented that it forms a tetramer in aqueous solution, whereas when solubilised in 1% Triton X-100 it remains monomeric. The secondary structure content of both monomeric and tetrameric K(V)1.1 N-terminal fragment has been estimated from FTIR and CD spectroscopy to be 20-25% alpha-helix, 20-25% beta-sheet, 20% turns and 30-40% random coil. Solubilisation of the protein in detergent is shown by hydrogen-deuterium exchange analysis to alter tertiary structure rather than secondary structure and this may be the determining factor in tetramerisation ability. Using molecular modelling we propose a supersecondary structure consisting of two structural domains.

Ultrastructural localization of Shaker-related potassium channel subunits and synapse-associated protein 90 to septate-like junctions in rat cerebellar Pinceaux.

The Pinceau is a paintbrush-like network of cerebellar basket cell axon branchlets embracing the initial segment of the Purkinje cell axon. Its electrical activity contributes to the control of the cerebellar cortical output through the Purkinje cell axon by generating an inhibitory field effect. In addition to the structural features of the Pinceau, its repertoire of voltage-gated ion channels is likely to be an important aspect of this function. Therefore, we investigated the fine structural distribution of voltage-activated potassium (Kv1.1, Kv1.2, Kv3.4) and sodium channel proteins in the Pinceau. The ultrastructural localization of potassium channel subunits was compared to the distribution of synapse-associated protein 90 (SAP90), a protein capable to induce in vitro clustering of Kv1 proteins. With an improved preembedding technique including ultrasmall gold particles, silver enhancement and gold toning, we could show that antibodies recognizing Kv1.1, Kv1.2 and SAP90 are predominantly localized to septate-like junctions, which connect the basket cell axonal branchlets. Kv3.4 immunoreactivity is not concentrated in junctional regions but uniformly distributed over the Pinceau and the pericellular basket surrounding the Purkinje cell soma. In contrast, voltage-activated sodium channels were not detected in the Pinceau, but localized to the Purkinje cell axon initial segment. The results suggest that Kv1.1 and Kv1.2 form heterooligomeric delayed rectifier type Kv channels, being colocalized to septate-like junctions by interaction with SAP90.

Polarized targeting of a shaker-like (A-type) K(+)-channel in the polarized epithelial cell line MDCK.

Functional Kv 1-4 channels were stably expressed in filter-grown MDCK cells which form a polarized epithelium with two distinct plasma membrane domains: a basolateral and an apical cell surface. The Shaker-related Kv 1-4 channels mediated in MDCK cells fast transient (A-type) voltage-activated outward currents having similar properties to the ones reported for Kv 1-4 in the Xenopus oocytes expression system. Immunoblot analysis with specific anti-Kv 1-4 antibodies showed that two Kv 1-4 protein forms are expressed in MDCK cells which most likely represent the glycosylated and non-glycosylated Kv 1-4 protein, respectively. Using immunocytochemistry and confocal microscopy we showed that the Kv 1-4 channels are specifically localized in the basolateral membranes of MDCK cells. Thus, the MDCK cells may provide an important model system to analyse the polarized transport of ion channels such as Kv 1-4, which are distinctly expressed in the mammalian central nervous system.

Kv beta 1 subunit binding specific for shaker-related potassium channel alpha subunits.

Voltage-activated potassium (Kv) channels from mammalian brain are hetero-oligomers containing alpha and beta subunits. Coexpression of Kv1 alpha and Kv beta 1 subunits confers rapid A-type inactivation on noninactivating potassium channels (delayed rectifiers) in expression systems in vitro. We have delineated a Kv1.5 aminoterminal region of up to 90 amino acids (residues 112-201) that is sufficient for interactions of Kv1.5 alpha and Kv beta 1 subunits. Within this region of the Kv1.5 amino terminus (residues 193-201), a Kv beta 1 interaction site necessary for Kv beta 1-mediated rapid inactivation of Kv1.5 currents was detected. This interaction site motif (FYE/QLGE/DEAM/L) is found exclusively in the Shaker-related subfamily (Kv1). The results show that hetero-oligomerization between alpha and Kv beta 1 subunits is restricted to Shaker-related potassium channel alpha subunits.

Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting subcellular locations and neuron-specific co-localizations in rat brain.

A large variety of potassium channels is involved in regulating integration and transmission of electrical signals in the nervous system. Different types of neurons, therefore, require specific patterns of potassium channel subunits expression and specific regulation of subunit coassembly into heteromultimeric channels, as well as subunit-specific sorting and segregation. This was investigated by studying in detail the expression of six different alpha-subunits of voltage-gated potassium channels in the rat hippocampus, cerebellum, olfactory bulb and spinal cord, combining in situ hybridization and immunocytochemistry. Specific polyclonal antibodies were prepared for five alpha-subunits (Kv1.1, Kv1.2, Kv1.3 Kv1.4, Kv1.6) of the Shaker-related subfamily of rat Kv channels, which encode delayed-rectifier type and rapidly inactivating A-type potassium channels. Their distribution was compared to that of an A-type potassium channel (Kv3.4), belonging to the Shaw-related subfamily of rat Kv channels. Our results show that these Kv channel alpha-subunits are differentially expressed in rat brain neurons. We did not observe in various neurons a stereotypical distribution of Kv channel alpha-subunits to dendritic and axonal compartments, but a complex differential subcellular subunit distribution. The different Kv channel subunits are targeted either to presynaptic or to postsynaptic domains, depending on neuronal cell type. Thus, distinct combinations of Kv1 alpha-subunits are co-localized in different neurons. The implications of these findings are that both differential expression and assembly as well as subcellular targeting of Kv channel alpha-subunits may contribute to Kv channel diversity and thereby to presynaptic and postsynaptic membrane excitability.

Antibodies specific for distinct Kv subunits unveil a heterooligomeric basis for subtypes of alpha-dendrotoxin-sensitive K+ channels in bovine brain.

The authentic subunit compositions of neuronal K+ channels purified from bovine brain were analyzed using a monoclonal antibody (mAb 5), reactive exclusively with the Kv1.2 subunit of the latter and polyclonal antibodies specific for fusion proteins containing C-terminal regions of four mammalian Kv proteins. Western blotting of the K+ channels isolated from several brain regions, employing the selective blocker alpha-dendrotoxin (alpha-DTX), revealed the presence in each of four different Kvs. Variable amounts of Kv1.1 and 1.4 subunits were observed in the K+ channels purified from cerebellum, corpus striatum, hippocampus, cerebral cortex, and brain stem; on the other hand, contents of Kv1.6 and 1.2 subunits appeared uniform throughout. Each Kv-specific antibody precipitated a different proportion (anti-Kv1.2 > 1.1 >> 1.6 > 1.4) of the channels detectable with radioiodinated alpha-DTX in every brain region, consistent with a widespread distribution of these oligomeric subtypes. Such heterooligomeric combinations were further documented by the lack of additivity upon their precipitation with a mixture of antibodies to Kv1.1 and Kv1.2; moreover, cross-blotting of the multimers precipitated by mAb 5 showed that they contain all four Kv proteins. Collectively, these findings demonstrate that subtypes of alpha-DTX-susceptible K+ channels are prevalent throughout mammalian brain which are composed of different Kv proteins assembled in complexes, shown previously to also contain auxiliary beta-subunits [Parcej, D. N., Scott, V. E. S., & Dolly, J.O. (1992) Biochemistry 31, 11084-11088].

Application of an ectopic expression system for the selection of protein-isoform-specific antibodies. The monoclonal antibody K1C3 is specific for the RCK1 potassium channel.

Monoclonal antibodies were raised against a fusion protein consisting of a fragment of 141 amino acids of the C-terminal region of the rat brain voltage-gated K(+)-channel protein (RCK1) and the lambda N protein (fusion protein I). Selection of K(+)-channel-specific hybridoma cell lines was performed by means of an ELISA employing a fusion protein consisting of the K(+)-channel-specific peptide sequence and glutathione S-transferase (fusion protein II). For final selection of RCK1 isoform-specific antibodies, a panel of Xenopus oocytes was employed, each injected with cRNA coding for a specific RCK isoform (RCK 1, 2, 4 or 5). Several days after injection, cryosections of embedded oocytes were obtained and were employed in immunohistochemical analysis of antibody binding. Of five hybridoma supernatants from stable growing hybridoma cell lines, selected by the fusion-protein ELISA, one monoclonal antibody (denoted K1C3) recognized exclusively the RCK1-protein isoform, with the other four exhibiting different levels of cross-reactivity with other K(+)-channel isoforms, or with unknown protein(s) of non-injected oocytes. The expression of the RCK1 protein in the postnatal brain was studied using, as far as we are aware, the first example of the application of such isoform-specific antibodies.

The Xenopus oocyte as an ectopic expression system for the selection of protein isoform-specific antibodies.

A panel of Xenopus oocytes, each injected with cRNA coding for one specific isoform of the rat brain RCK family of voltage gated potassium channel proteins, was employed to screen for isoform-specific monoclonal antibodies. Several days after injection, cryosections of embedded oocytes were produced and were employed in immunohistochemical analysis of antibody binding. Of the advantageous properties of the assay, it employs the native antigen, it can be applied to homooligomeric and heterooligomeric proteins, and cryosections of the same batch can be stored frozen for later tests. The method may be advantageous also for the selection of isoform-specific antibodies of other protein families.

Heteromultimeric channels formed by rat brain potassium-channel proteins.

An important step towards understanding the molecular basis of the functional diversity of voltage-gated K+ channels in the mammalian brain has been the discovery of a family of genes encoding rat brain K+ channel-forming (RCK) proteins. All species of these RCK proteins form homomultimeric voltage-gated K+ channels with distinct functional characteristics in Xenopus laevis oocytes following injection of the respective cRNAs. RCK-specific mRNAs are coexpressed in several regions of the brain, suggesting that RCK proteins also assemble into heteromultimeric K+ channels. In addition expression experiments with fractionated poly(A)+ mRNA have suggested that heteromultimeric K+ channels may occur in mammalian brain. We report here that heteromultimeric K+ channels composed of two different RCK proteins (RCK1 and RCK4) assemble after cotransfection of HeLa cells with the corresponding cDNAs and after coinjection of the corresponding cRNAs into Xenopus oocytes. The heteromultimeric RCK1, 4 channel mediates a transient potassium outward current, similar to the RCK4 channel but inactivates more slowly, has a larger conductance and is more sensitive to block by dendrotoxin and tetraethylammonium chloride.

The interference of truncated with normal potassium channel subunits leads to abnormal behaviour in transgenic Drosophila melanogaster.

The Shaker locus of Drosophila melanogaster encodes a family of A-type potassium channel subunits. Shaker mutants behave as antimorphs in gene dosage tests. This behaviour is due to the production of truncated A-channel subunits. We propose that they interfere with the function of their normal counterpart by forming multimeric A-channel structures. This hypothesis was tested by constructing transgenic flies carrying a heat-inducible gene encoding a truncated A-type potassium channel subunit together with a normal wild type doses of A-type potassium channel subunits. The altered subunit leads at larval, pupal or adult stages to the transformation of wild type into Shaker flies. The transformed flies exhibited a heat-inducible abnormal leg shaking behaviour and a heat-inducible facilitated neurotransmitter release at larval neuromuscular junctions. By the overexpression of an aberrant A-channel subunit the normal behaviour of transgenic D. melanogaster can be altered in a predictable way.