Isoform-specific insulin receptor signaling involves different plasma membrane domains

In pancreatic β-cells, insulin selectively up-regulates the transcription of its own gene and that of the glucokinase gene by signaling through the two isoforms of the insulin receptor, i.e., A-type (Ex11−) and B-type (Ex11+), using different signaling pathways. However, the molecular mechanism(s) that allows the discrete activation of signaling cascades via the two receptor isoforms remains unclear. Here we show that activation of the insulin promoter via A-type and of the glucokinase promoter via B-type insulin receptor is not dependent on receptor isoform–specific differences in internalization but on the different localization of the receptor types in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12–amino acid string encoded by exon 11, which acts as a sorting signal rather than as a physical spacer. Moreover, our data suggest that selective activation of the insulin and glucokinase promoters occurs by signaling from noncaveolae lipid rafts that are differently sensitive toward cholesterol depletion

Ectopic leptin production by intraocular pancreatic islet organoids ameliorates the metabolic phenotype of ob/ob mice

The pancreatic islets of Langerhans consist of endocrine cells that secrete peptide hormones into the blood circulation in response to metabolic stimuli. When transplanted into the anterior chamber of the eye (ACE), pancreatic islets engraft and maintain morphological features of native islets as well as islet-specific vascularization and innervation patterns. In sufficient amounts, intraocular islets are able to maintain glucose homeostasis in diabetic mice. Islet organoids (pseudo-islets), which are formed by self-reassembly of islet cells following disaggregation and genetic manipulation, behave similarly to native islets. Here, we tested the hypothesis that genetically engineered intraocular islet organoids can serve as production sites for leptin. To test this hypothesis, we chose the leptin-deficient ob/ob mouse as a model system, which becomes severely obese, hyperinsulinemic, hyperglycemic, and insulin resistant. We generated a Tet-OFF-based beta-cell-specific adenoviral expression construct for mouse leptin, which allowed efficient transduction of native beta-cells, optical monitoring of leptin expression by co-expressed fluorescent proteins, and the possibility to switch-off leptin expression by treatment with doxycycline. Intraocular transplantation of islet organoids formed from transduced islet cells, which lack functional leptin receptors, to ob/ob mice allowed optical monitoring of leptin expression and ameliorated their metabolic phenotype by improving bodyweight, glucose tolerance, serum insulin, and C-peptide levels

Inward and outward currents of native and cloned K(ATP) channels (Kir6.2/SUR1) share single-channel kinetic properties

BACKGROUND: The ATP-sensitive K(+) (K(ATP)) channel is found in a variety of tissues extending from the heart and vascular smooth muscles to the endocrine pancreas and brain. Common to all K(ATP) channels is the pore-forming subunit Kir6.x, a member of the family of small inwardly rectifying K(+) channels, and the regulatory subunit sulfonylurea receptor (SURx). In insulin secreting β-cells in the endocrine part of the pancreas, where the channel is best studied, the K(ATP) channel consists of Kir6.2 and SUR1. Under physiological conditions, the K(ATP) channel current flow is outward at membrane potentials more positive than the K(+) equilibrium potential around −80 mV. However, K(ATP) channel kinetics have been extensively investigated for inward currents and the single-channel kinetic model is based on this type of recording, whereas only a limited amount of work has focused on outward current kinetics. METHODS: We have estimated the kinetic properties of both native and cloned K(ATP) channels under varying ionic gradients and membrane potentials using the patch-clamp technique. RESULTS: Analyses of outward currents in K(ATP) and cloned Kir6.2ΔC26 channels, alone or co-expressed with SUR1, show openings that are not grouped in bursts as seen for inward currents. Burst duration for inward current corresponds well to open time for outward current. CONCLUSIONS: Outward K(ATP) channel currents are not grouped in bursts regardless of membrane potential, and channel open time for outward currents corresponds to burst duration for inward currents

Human islet microtissues as an in vitro and an in vivo model system for diabetes

Loss of pancreatic β-cell function is a critical event in the pathophysiology of type 2 diabetes. However, studies of its underlying mechanisms as well as the discovery of novel targets and therapies have been hindered due to limitations in available experimental models. In this study we exploited the stable viability and function of standardized human islet microtissues to develop a disease-relevant, scalable, and reproducible model of β-cell dysfunction by exposing them to long-term glucotoxicity and glucolipotoxicity. Moreover, by establishing a method for highly-efficient and homogeneous viral transduction, we were able to monitor the loss of functional β-cell mass in vivo by transplanting reporter human islet microtissues into the anterior chamber of the eye of immune-deficient mice exposed to a diabetogenic diet for 12 weeks. This newly developed in vitro model as well as the described in vivo methodology represent a new set of tools that will facilitate the study of β-cell failure in type 2 diabetes and would accelerate the discovery of novel therapeutic agents

Insulin signaling in the pancreatic beta-cell

The appropriate function of insulin-producing pancreatic beta-cells is crucial for the regulation of glucose homeostasis, and its impairment leads to diabetes mellitus, the most common metabolic disorder in man. In addition to glucose, the major nutrient factor, inputs from the nervous system, humoral components, and cell-cell communication within the islet of Langerhans act together to guarantee the release of appropriate amounts of insulin in response to changes in blood glucose levels. Data obtained within the past decade in several laboratories have revitalized controversy over the autocrine feedback action of secreted insulin on beta-cell function. Although insulin historically has been suggested to exert a negative effect on beta-cells, recent data provide evidence for a positive role of insulin in transcription, translation, ion flux, insulin secretion, proliferation, and beta-cell survival. Current insights on the role of insulin on pancreatic beta-cell function are discussed

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

AbstractTranscriptional 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 betwee 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

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 β cell promotes insulin biosynthesis by enhancing insulin gene transcription in an autocrine manner. We show that secreted insulin acts via β-cell insulin receptors and up-regulates insulin gene transcription by signaling through the IRS-2/PI-3 kinase/p70 s6k and CaM kinase pathways

Glucose-stimulated Insulin Biosynthesis Depends on Insulin-stimulated Insulin Gene Transcription

Glucose stimulation of pancreatic β-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