Non-invasive in vivo imaging of pancreatic ß-cell function and survival - a perspective.

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/ß-cells by techniques, such as magnetic resonance imaging and positron emission tomography, and present an experimental platform, which allows in vivo imaging of pancreatic ß-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 ß-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.

Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: indications for a long-chain acyl-CoA ester binding motif.

AIMS/HYPOTHESIS: The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K(ATP) channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. MATERIALS AND METHODS: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. RESULTS: Oocytes expressing Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K(ATP) channel and Kir6.2DeltaC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. CONCLUSIONS/INTERPRETATION: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.

Short-term regulation of insulin gene transcription.

Short-term regulation of insulin gene transcription is still a matter of debate. However, an increasing body of evidence shows that insulin gene transcription is affected by signals, such as incretins, glucose metabolites, intracellular Ca2+, and by insulin secreted from pancreatic beta-cells, all supporting the concept of an immediate response resulting in insulin gene transcription following food-uptake. The present review aims to summarize the current view on the mechanisms underlying the up-regulation of insulin gene transcription in response to glucose, the major nutrient factor in insulin secretion and biosynthesis.

Selective insulin signaling through A and B insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic beta cells.

Insulin signaling is mediated by a complex network of diverging and converging pathways, with alternative proteins and isoforms at almost every step in the process. We show here that insulin activates the transcription of its own gene and that of the beta cell glucokinase gene (betaGK) by different mechanisms. Whereas insulin gene transcription is promoted by signaling through insulin receptor A type (Ex11-), PI3K class Ia, and p70s6k, insulin stimulates the betaGK gene by signaling via insulin receptor B type (Ex11+), PI3K class II-like activity, and PKB (c-Akt). Our data provide evidence for selectivity in insulin action via the two isoforms of the insulin receptor, the molecular basis being preferential signaling through different PI3K and protein kinases.

Online monitoring of stimulus-induced gene expression in pancreatic beta-cells.

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.

Glucose-stimulated insulin biosynthesis depends on insulin-stimulated insulin gene transcription.

Glucose stimulation of pancreatic beta-cells leads to insulin secretion as well as up-regulation of insulin biosynthesis. The acute elevation in pro-insulin levels is thought to be exclusively because of the activation of translation of pre-existing prepro-insulin mRNA. Glucose-stimulated insulin gene transcription is believed to be a long term effect and should therefore not contribute to the acute elevation in pro-insulin levels. We have recently shown that glucose activates insulin gene transcription within minutes and that secreted insulin is one of the key factors triggering this process in an autocrine manner. We now provide evidence that 50% of the glucose-stimulated, acute pro-insulin biosynthesis within 30 min results from up-regulated insulin gene transcription. Our data led us to propose that glucose elevates pro-insulin levels by stimulating both transcriptional and post-transcriptional/post-translational events to an equal extent. Whereas the stimulatory effect on transcription is mediated by insulin secreted in response to glucose, glucose directly stimulates the post-transcriptional/post-translational processes.

Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1.

Pancreatic duodenal homeobox-containing transcription factor 1 (PDX-1) plays a crucial role in pancreas development and beta-cell gene regulation. Absence of PDX-1 leads to pancreas agenesis and its malfunction causes MODY4 diabetes mellitus. PDX-1 has been suggested to be involved in the glucose-dependent regulation of insulin gene transcription. Whereas DNA-binding and transactivation domains of PDX-1 are in the process of being characterized, protein sequences responsible for its nuclear translocation remain unknown. By combining site-directed mutagenesis of putative phosphorylation sites and nuclear localization signal (NLS) motifs with on-line monitoring of GFP-tagged PDX-1 translocation, we demonstrate that the NLS motif RRMKWKK is necessary and in conjunction with the integrity of the 'helix 3' domain of the PDX-1 homeodomain is sufficient for the nuclear import of PDX-1. Furthermore, we show that there is no glucose-dependent cytoplasmic-nuclear cycling of PDX-1.

Syntaxin 1 interacts with the L(D) subtype of voltage-gated Ca(2+) channels in pancreatic beta cells.

Interaction of syntaxin 1 with the alpha(1D) subunit of the voltage-gated L type Ca(2+) channel was investigated in the pancreatic beta cell. Coexpression of the enhanced green fluorescent protein-linked alpha(1D) subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the alpha(1D) subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the alpha(1D) subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca(2+) channel activity because there was a faster Ca(2+) current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K(+) or exposure to high Ca(2+). Exchanging Ca(2+) for Ba(2+) abolished the effect of antisyntaxin 1 on both Ca(2+) channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca(2+)-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure-function relationship between the alpha(1D) subunit of the L type Ca(2+) channel and the exocytotic machinery in the pancreatic beta cell.

Long chain coenzyme A esters activate the pore-forming subunit (Kir6. 2) of the ATP-regulated potassium channel.

The ATP-dependent potassium (KATP) channel in the pancreatic beta-cell is a complex of two proteins, the pore-forming Kir6.2 and the sulfonylurea receptor type 1 (SUR1). Both subunits are required for functional KATP channels because expression of Kir6.2 alone does not result in measurable currents. However, truncation of the last 26 or 36 amino acids of the C terminus of Kir6.2 enables functional expression of the pore-forming protein in the absence of SUR1. Thus, by using the truncated form of Kir6.2, expressed in the absence and presence of SUR1, it has been shown that the site at which ATP mediates channel inhibition is likely to be situated on Kir6.2. We have now examined the effects of long chain acyl-CoA (LC-CoA) esters on the C-terminally truncated mouse Kir6.2Delta365-390 (Kir6. 2DeltaC26) in inside-out patches isolated from Xenopus laevis oocytes. LC-CoA esters, saturated (C14:0, C16:0) and unsaturated (C18:1), increased Kir6.2DeltaC26 currents, whereas short and medium chain CoA esters (C3:0, C8:0, C12:0) were unable to affect channel activity. The LC-CoA esters were also able to counteract the blocking effect of ATP on Kir6.2DeltaC26. The stimulatory effect of the esters could be explained by the induction of a prolonged open state of Kir6.2DeltaC26. In the presence of the esters, channel open time was increased approximately 3-fold, which is identical to what was obtained in the native mouse KATP channel. Coexpression of SUR1 together with Kir6.2DeltaC26 did not further increase the ability of LC-CoA esters to stimulate channel activity. We conclude that Kir6.2 is the primary target for LC-CoA esters to activate the KATP channel and that the esters are likely to induce a conformational change by a direct interference with the pore-forming subunit, leading to openings of long duration.

Short-term regulation of insulin gene transcription by glucose.

Whereas short-term regulation of insulin biosynthesis at the level of translation is well accepted, glucose-dependent transcriptional control is still believed to be a long-term effect occurring after more than 2 hr of glucose stimulation. Because pancreatic beta cells are exposed to elevated glucose levels for minutes rather than hours after food uptake, we hypothesized the existence of a short-term transcriptional control. By studying the dynamics of newly synthesized (prepro)insulin RNA and by employing on-line monitoring of gene expression in single, insulin-producing cells, we were able to provide convincing evidence that insulin gene transcription indeed is affected by glucose within minutes. Exposure of insulinoma cells and isolated pancreatic islets to elevated glucose for only 15 min resulted in a 2- to 5-fold elevation in (prepro)insulin mRNA levels within 60-90 min. Similarly, insulin promoter-driven green fluorescent protein expression in single insulin-producing cells was significantly enhanced after transient glucose stimulation. Thus, short-term signaling, such as that involved in insulin secretion, also may regulate insulin gene transcription.

Exocytosis of insulin promotes insulin gene transcription via the insulin receptor/PI-3 kinase/p70 s6 kinase and CaM kinase pathways.

The control of glucose homeostasis by insulin requires, in addition to the glucose-induced insulin release, a highly dynamic control of insulin biosynthesis. Although elevated glucose concentrations have been shown to trigger insulin biosynthesis at the levels of transcription and translation, the molecular mechanisms underlying the immediate transcriptional control are poorly understood. By investigating signal transduction pathways involved in the "glucose-dependent" transcriptional control, thereby analyzing endogenous (prepro)insulin mRNA levels and monitoring on-line insulin promoter-driven GFP expression, we provide, for the first time, evidence that physiologically stimulated insulin secretion from the pancreatic beta cell promotes insulin biosynthesis by enhancing insulin gene transcription in an autocrine manner. We show that secreted insulin acts via beta-cell insulin receptors and up-regulates insulin gene transcription by signaling through the IRS-2/PI-3 kinase/p70 s6k and CaM kinase pathways.

Dual function of the intron of the rat insulin I gene in regulation of gene expression.

Since the short intron in the 5'-untranslated region (5'-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5'-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5'-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins11VS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides -87 and +110 showed a reduction of expression by 30%. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.

Functional analysis of a newly identified TAAT-box of the rat insulin-II gene promoter.

Transcriptional regulation of insulin gene expression is achieved by an interplay of tissue-specific and ubiquitous cis- and trans-acting elements. E-box like motifs and TAAT-motifs were shown to play a crucial role in initiating insulin gene transcription. Studying the AT-rich region of the rat insulin-II promoter between nucleotides -212 and -196, we observed a base difference at -211, an adenosine instead of a cytidine, compared to the previously reported sequence (EMBL Accession No. J00748). Sequence analysis of promoter fragments from different rat strains showed that adenosine at position -211 represents the wild type (EMBL Accession No. X82162). This base exchange leads to the formation of an additional TAAT-motif, i.e. TAAT3, at the complementary DNA strand directly upstream of the previously studied TAAT2 motif, formerly named CT-2. Here we show that the newly identified motif TAAT3 is involved in (i) transcriptional control in vivo, (ii) in vitro DNA/protein interactions, and that (iii) TAAT1, TAAT2 and TAAT3 are binding sites for the homeodomain-containing factor IPF-1.

Functional analysis of DNA-elements involved in transcriptional control of the human glucose transporter 2 (GLUT 2) gene in the insulin-producing cell line beta TC-3.

The uptake of glucose into pancreatic beta cells as a 'non-rate-limiting-step' is guaranteed by the expression and action of the high-Km glucose transporter 2 (GLUT 2). This transporter is not saturated by physiological plasma glucose levels and hence functions as a "glucose sensor/glucoreceptor". Here we describe DNA-elements of the human GLUT 2 gene promoter which contribute to transcriptional control in the insulin-producing cell line beta TC-3. Nested 5'-as well as 3'-deletions of a DNA-fragment containing up to 1245 bp of the 5'-flanking region and up to 308 bp of the first exon of the human GLUT 2 gene were investigated for their ability to control the expression of a CAT reporter gene in beta TC-3 cells. For tissue-specific transcriptional control 5'-deletional analysis revealed that the region -220/+309 was sufficient. Truncation from the 3'-end from nucleotide +308 to +204 led to a threefold drop in CAT expression. In vitro DNase I footprinting analysis was performed to delineate cis-elements within the region -220/+1. Five specifically protected areas could be defined.

CT-boxes are involved in control of the rat insulin II gene expression.

Expression of the rat insulin II gene is controlled mainly at the level of transcription initiation by multiple factors binding to specific cis-acting DNA-elements in the regulatory region. We have shown that two elements (CT-motifs) located between nucleotides -83 and -76 (CT-1) and -204 and -197 (CT-2) are involved in transcriptional regulation in the insulin-producing cell line HIT M2.2.2. Transient expression analysis of 5'-deletion as well as block replacement mutants revealed that CT-1 and CT-2 are mutational sensitive. Gel mobility shift assays showed that both motifs bind similar nuclear factors. Our results suggest the involvement of a third CT-motif located directly upstream of CT-2 on the complementary strand.

The role of the proximal CTAAT-box of the rat glucokinase upstream promoter in transcriptional control in insulin-producing cells.

Sequence analysis of the 5'flanking region of the beta-cell specific transcription unit of the rat glucokinase gene (r beta GK) revealed the presence of sequence motifs very similar to the IEB-(Far)-box and a CT-motif which play a crucial role in transcriptional control of insulin genes. 5'deletional analysis of the r beta GK proximal promoter element (localized between nucleotides -278 and -49) as well as site directed mutagenesis showed that both motifs are mutationally sensitive and contribute to transcriptional control in HIT M2.2.2 cells. The combination of the IEB-(Far)-like motif with the CT-box was unable to form a "mini-enhancer" similar to the Far-FLAT-element of the rat insulin I gene promoter but rather functions as a beta-cell specific control element in r beta GK expression. Electrophoretic mobility shift assays (EMSAs) and competition studies using oligonucleotides containing CT-motifs of rat insulin genes promoters, human insulin gene promoter, and rat amylin gene promoter showed similar binding patterns with nuclear extracts isolated from insulin-producing cell lines. These studies indicate that CT-motifs of rat glucokinase, insulin, and amylin gene promoters may bind similar--probably identical--nuclear factor(s) and may play a central role in the coordinated expression of these genes in insulin-producing cells.

Positive and negative regulatory elements are involved in transcriptional control of the rat glucokinase gene in the insulin producing cell line HIT M2.2.2.

Nested deletion mutants of the 5' flanking region of the beta-cell transcription unit of the rat glucokinase gene (r beta GK) were fused to the CAT-reporter gene. Transient expression studies in HIT M2.2.2 and BHK21 cells revealed a distal (upstream of -359) and a proximal promoter region (between -278/-49) harbouring positive and negative regulatory elements. DNaseI footprinting revealed three protected areas between nucleotides -190 and -60. DNA-elements playing a crucial role in transcriptional control of the insulin genes (IEB- and CT-motifs) have been detected within the proximal promoter region and contribute to beta-cell specific gene regulation. 3' deletion analysis revealed that DNA-elements located downstream from transcription initiation sites (up to +123) contribute to transcriptional regulation.