Search

Insulin and [Zn.sup.2+] enjoy a multivalent relationship. [Zn.sup.2+] binds insulin in pancreatic β cells to form crystalline aggregates in dense core vesicles (DCVs), which are released in response to physiological [...]

Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ER[alpha] also enhances insulin synthesis in cultured islets. Whether ERa stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERa knockout mouse (PER[alpha][KO.sup.-/-]) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and [beta] cells. Whereas 17[beta]-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERcdox/lox mice, these E2 effects are abolished in PER[alpha][KO.sup.-/-] mice. We find that E2-activated ER[alpha] increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ER[alpha] eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ER[alpha] interacts with the tyrosine kinase Src, which activates extracellular signal-regulated [kinases.sub.1/2], to increase nuclear localization and binding to the insulin promoter of the transcription factor NeuroD1. This study supports the importance of ERa in [beta] cells as a regulator of insulin synthesis in vivo. diabetes | islet doi/ 10.1073/pnas.0914501107

The C-terminal segment of the human insulin receptor a-chain (designated [alpha]CT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-cross-linking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The [alpha]CT segment is now resolved as being engaged with the central [beta]-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is a-helical in conformation and extends 11 residues N-terminal of the classical [alpha]CT segment boundary originally defined by peptide mapping. This tandem structural element ([alpha]CT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant [alpha]CT peptides binding to an insulin minireceptor, leads to the conclusion that putative 'insulin-mimetic' peptides in the literature act at least in part as mimics of the [alpha]CT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the [alpha]CT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate [alpha]-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus. diabetes | IGF-1 receptor | insulin binding | photo-cross-linking | protein crystallography doi/ 10.1073/pnas.1001813107

Quantification of insulin release from pancreatic islets of Langerhans is of interest for diabetes research. Typical insulin secretion experiments are performed using offline techniques that are expensive, slow, have low-throughput, and require multiple islets. We have developed a microfluidic device for high-throughput, automated, and online monitoring of insulin secretion from individual islets in parallel. This chip consists of 15 channel networks each capable of superfusing a single islet and mixing superfusate from each islet online with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for a competitive immunoassay. The resulting continuous reaction streams are periodically injected onto parallel electrophoresis channels where the mixtures are separated. The resulting traces are used to quantify relative insulin released from islets. Serial immunoassays were performed at 10 s intervals on all 15 channels, corresponding to 5400 immunoassays per hour, to create temporally resolved insulin release profiles that captured single islet secretion dynamics. The chip was used to demonstrate that free fatty acid induced lipotoxicity in islets eliminates pulsatile insulin secretion.

Renal tubule epithelial cells express the insulin receptor (IR); however, their value has not been firmly established. We generated mice with renal epithelial cell-specific knockout of the IR by Cre-recombinase-loxP recombination using a kidney-specific (Ksp) cadherin promoter. KO mice expressed significantly lower levels of IR mRNA and protein in kidney cortex (49-56% of the WT) and medulla (32-47%) homogenates. Immunofluorescence showed the greatest relative reduction in the thick ascending limb and collecting duct cell types. Body weight, kidney weight, and food and water intakes were not different from WT littermates. However, KO mice had significantly increased basal systolic blood pressure (BP, 15 mm Hg higher) as measured by radiotelemetry. In response to a volume load by gavage (20 ml/kg of body weight, 0.9% NaCl, 15% dextrose), KO mice had impaired natriuresis (37 [+ or -] 10 versus 99 [+ or -] 9 mmol of [Na.sup.+] per 2 h in WT). Furthermore, volume load led to a sustained increase in BP in KO mice only. In contrast, insulin administration i.p. (0.5 units/kg of body weight) resulted in a significant fall in BP in WT, but not in KO mice. To test the role of reduced renal nitric oxide (NO) production in these responses, basal urinary nitrates plus nitrites excretion (UNOx) was measured and found to be 61% lower in KO vs. WT mice. Furthermore, acute insulin increased UNOx by 202% in the WT, relative to a significantly blunted rise (67%) in KO animals. These results illuminate a previously uncharacterized role for renal IR to reduce BP and facilitate sodium and water excretion, possibly via NO production. diabetes | metabolic syndrome | natriuresis | volume expansion

The insulin signaling pathway is of pivotal importance in metabolic diseases, such as diabetes, and in cellular processes, such as aging. Insulin activates a tyrosine phosphorylation cascade that branches to create a complex network affecting multiple biological processes. To understand the full spectrum of the tyrosine phosphorylation cascade, we have defined the tyrosine-phosphoproteome of the insulin signaling pathway, using high resolution mass spectrometry in combination with phosphotyrosine immunoprecipitation and stable isotope labeling by amino acids in cell culture (SILAC) in differentiated brown adipocytes. Of 40 identified insulin-induced effectors, 7 have not previously been described in insulin signaling, including SDR, PKC[delta] binding protein, LRP-6, and PISP/ PDZK11, a potential calcium ATPase binding protein. A proteomic interaction screen with PISP/PDZK11 identified the calcium transporting ATPase SERCA2, supporting a connection to calcium signaling. The combination of quantitative phosphoproteomics with cell culture models provides a powerful strategy to dissect the insulin signaling pathways in intact cells. diabetes | insulin action | tyrosine phosphorylation

A simulation model of the glucose-insulin system in the postprandial state can be useful in several circumstances, including testing of glucose sensors, insulin infusion algorithms and decision support systems for diabetes. Here, we present a new simulation model in normal humans that describes the physiological events that occur after a meal, by employing the quantitative knowledge that has become available in recent years. Model parameters were set to fit the mean data of a large normal subject database that underwent a triple tracer meal protocol which provided quasi-model-independent estimates of major glucose and insulin fluxes, e.g., meal rate of appearance, endogenous glucose production, utilization of glucose, insulin secretion. By decomposing the system into subsystems, we have developed parametric models of each subsystem by using a forcing function strategy. Model results are shown in describing both a single meal and normal daily life (breakfast, lunch, dinner) in normal. The same strategy is also applied on a smaller database for extending the model to type 2 diabetes. Index Terms--Artificial pancreas, diabetes, glucose homeostasis, glucose production, glucose sensors, glucose utilization, insulin infusion system, insulin secretion, kinetics, physiological control.