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Ghrelin is a novel 28-amino acid gut-brain peptide, which was first isolated in the rat stomach. This study examined the effect of ghrelin on insulin secretion from the isolated pancreas of normal and diabetic rats. Diabetes was induced by a single dose of streptozotocin. Four weeks after the induction of diabetes, pancreatic tissue fragments of normal and diabetic rats were treated with different concentrations (10(-12), 10(-9) and 10(-6) M) of ghrelin. Ghrelin evoked large and significant increases in insulin secretion from the pancreas of both normal and diabetic rats. In the pancreas of normal rats, diltiazem (calcium channel antagonist) or a combination of atropine (muscarinic cholinergic receptor antagonist), propranolol (beta-adrenergic receptor antagonist) and yohimbine (alpha2-adrenergic receptor antagonist) significantly reduced the stimulatory effect of ghrelin on insulin secretion. Diltiazem and yohimbine failed to inhibit ghrelin-evoked insulin release in diabetic rat pancreas. Ghrelin-immunoreactivity cells was observed in 2.6% and 3.8% of the total cell population in the islet of Langerhans of normal and diabetic rats, respectively.

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.

Current methods to promote growth of cultured neurons use two-dimensional (2D) glass or polystyrene surfaces coated with a charged molecule (e.g. poly-L-lysine (PLL)) or an isolated extracellular matrix (ECM) protein (e.g. laminin-1). However, these 2D surfaces represent a poor topological approximation of the three-dimensional (3D) architecture of the assembled ECM that regulates neuronal growth in vivo. Here we report on the development of a new 3D synthetic nanofibrillar surface for the culture of neurons. This nanofibrillar surface is composed of polyamide nanofibers whose organization mimics the porosity and geometry of the ECM. Neuronal adhesion and neurite outgrowth from cerebellar granule, cerebral cortical, hippocampal, motor, and dorsal root ganglion neurons were similar on nanofibers and PLL-coated glass coverslips; however, neurite generation was increased. Moreover, covalent modification of the nanofibers with neuroactive peptides derived from human tenascin-C significantly enhanced the ability of the nanofibers to facilitate neuronal attachment, neurite generation, and neurite extension in vitro. Hence the 3D nanofibrillar surface provides a physically and chemically stabile cell culture surface for neurons and, potentially, an exciting new opportunity for the development of peptide-modified matrices for use in strategies designed to encourage axonal regrowth following central nervous system injury.

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Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates.

This study examined the changes occurring in the pattern of distribution and expression of neuronal nitric oxide synthase (nNOS)-positive nerves in the gastroduodenal tract of streptozotocin-induced diabetic rats. The ganglion cells of the myenteric plexus of the gastric antrum of normal rats contain nNOS. We also observed nNOS-positive neurons and fibres in the myenteric plexus of the duodenum of normal rats. After the onset of diabetes, the number and intensity of staining of nNOS-positive nerve profiles in the gastric antrum and duodenum did not change significantly. However, Western blotting showed a significant increase in the expression of nNOS after the onset of diabetes. In conclusion, diabetes of 4 and 32 weeks duration induced an increase in the tissue content of nNOS in the gastroduodenum of rat. The increase in the level of nNOS in the gastroduodenum of diabetic rats may explain why impaired gastric emptying is common in patients with diabetes.

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