Glucokinase intrinsically regulates glucose sensing and glucagon secretion in pancreatic alpha cells.

The secretion of glucagon by pancreatic alpha cells is regulated by a number of external and intrinsic factors. While the electrophysiological processes linking a lowering of glucose concentrations to an increased glucagon release are well characterized, the evidence for the identity and function of the glucose sensor is still incomplete. In the present study we aimed to address two unsolved problems: (1) do individual alpha cells have the intrinsic capability to regulate glucagon secretion by glucose, and (2) is glucokinase the alpha cell glucose sensor in this scenario. Single cell RT-PCR was used to confirm that glucokinase is the main glucose-phosphorylating enzyme expressed in rat pancreatic alpha cells. Modulation of glucokinase activity by pharmacological activators and inhibitors led to a lowering or an increase of the glucose threshold of glucagon release from single alpha cells, measured by TIRF microscopy, respectively. Knockdown of glucokinase expression resulted in a loss of glucose control of glucagon secretion. Taken together this study provides evidence for a crucial role of glucokinase in intrinsic glucose regulation of glucagon release in rat alpha cells.

Aptamer-functionalized porous phospholipid nanoshells for direct measurement of Hg(2+) in urine.

A porous phospholipid nanoshell (PPN) sensor functionalized with a specific aptamer sensor agent was prepared for rapid detection of Hg(2+) in human urine with minimal sample preparation. Aptamer sensors provide an important class of optical transducers that can be readily and reproducibly synthesized. A key limitation of aptamer sensors, and many other optical sensors, is the potential of biofouling or biodegradation when used in complex biological matrices such as serum or urine, particularly when high levels of nucleases are present. We prepared Hg(2+)-responsive, PPN-encapsulated aptamer sensors that overcome these limitations. PPNs provide a protective barrier to encapsulate the aptamer sensor in an aqueous environment free of diffusional restrictions encountered with many polymer nanomaterials. The unique porous properties of the PPN membrane enable ready and rapid transfer of small molecular weight ions and molecules into the sensor interior while minimizing the macromolecular interactions between the transducer and degradants or interferents in the exterior milieu. Using Hg(2+)-responsive, PPN-encapsulated aptamer sensors, we were able to detect sub-100 ppb (chronic threshold limit from urine test) Hg(2+) in human urine with no sample preparation, whereas free aptamer sensors yielded inaccurate results due to interferences from the matrix. The PPN architecture provides a new platform for construction of aptamer-functionalized sensors that target low molecular weight species in complex matrices, beyond the Hg(2+) demonstrated here.

Glucagon regulates its own synthesis by autocrine signaling.

Peptide hormones are powerful regulators of various biological processes. To guarantee continuous availability and function, peptide hormone secretion must be tightly coupled to its biosynthesis. A simple but efficient way to provide such regulation is through an autocrine feedback mechanism in which the secreted hormone is "sensed" by its respective receptor and initiates synthesis at the level of transcription and/or translation. Such a secretion-biosynthesis coupling has been demonstrated for insulin; however, because of insulin's unique role as the sole blood glucose-decreasing peptide hormone, this coupling is considered an exception rather than a more generally used mechanism. Here we provide evidence of a secretion-biosynthesis coupling for glucagon, one of several peptide hormones that increase blood glucose levels. We show that glucagon, secreted by the pancreatic α cell, up-regulates the expression of its own gene by signaling through the glucagon receptor, PKC, and PKA, supporting the more general applicability of an autocrine feedback mechanism in regulation of peptide hormone synthesis.

Determination of pore sizes and relative porosity in porous nanoshell architectures using dextran retention with single monomer resolution and proton permeation.

Unilamellar phospholipid vesicles prepared using the polymerizable lipid bis-sorbylphosphatidylcholine (bis-SorbPC) yield three-dimensional nanoarchitectures that are highly permeable to small molecules. The resulting porous phospholipid nanoshells (PPNs) are potentially useful for a range of biomedical applications including nanosensors and nanodelivery vehicles for cellular assays and manipulations. The uniformity and size distribution of the pores, key properties for sensor design and utilization, have not previously been reported. Fluorophore-assisted carbohydrate electrophoresis (FACE) was utilized to assess the nominal molecular weight cutoff limit (NMCL) of the PPN via analysis of retained dextran with single monomer resolution. The NMCL of PPNs prepared from pure bis-SorbPC was equivalent to a 1800 Da linear dextran, corresponding to a maximum pore diameter of 2.6 nm. Further investigation of PPNs prepared using binary mixtures of bis-SorbPC and dioleoylphosphatidylcholine (DOPC) revealed a similar NMCL when the bis-SorbPC content exceeded 30 mol %, whereas different size-dependent permeation was observed below this composition. Below 30 mol % bis-SorbPC, dextran retention provided insufficient mass resolution (162 Da) to observe porosity on the experimental time scale; however, proton permeability showed a marked enhancement for bis-SorbPC ≥ 10 mol %. Combined, these data suggest that the NMCL for native pores in bis-SorbPC PPNs results from an inherent property within the lipid assembly that can be partially disrupted by dilution of bis-SorbPC below a critical value for domain formation. Additionally, the analytical method described herein should prove useful for the challenging task of elucidating porosity in a range of three-dimensional nanomaterials.

Analysis of protein kinase A activity in insulin-secreting cells using a cell-penetrating protein substrate and capillary electrophoresis.

A cell-penetrating, fluorescent protein substrate was developed to monitor intracellular protein kinase A (PKA) activity in cells without the need for cellular transfection. The PKA substrate (PKAS) was prepared with a 6xhistidine purification tag, an enhanced green fluorescent protein (EGFP) reporter, an HIV-TAT protein transduction domain for cellular translocation and a pentaphosphorylation motif specific for PKA. PKAS was expressed in Escherichia coli and purified by metal affinity chromatography. Incubation of PKAS in the extracellular media facilitated translocation into the intracellular milieu in HeLa cells, betaTC-3 cells and pancreatic islets with minimal toxicity in a time and concentration dependent manner. Upon cellular loading, glucose-dependent phosphorylation of PKAS was observed in both betaTC-3 cells and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 +/- 6%) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 +/- 4%) in betaTC-3 cells was observed at 3 mM glucose indicating a left-shifted glucose sensitivity. Increased PKAS phosphorylation was observed in the presence of PKA stimulators forskolin and 8-Br-cAMP (33% and 16%, respectively), with corresponding decreases in PKAS phosphorylation observed in the presence of PKA inhibitors staurosporine and H-89 (40% and 54%, respectively).