Pancreatic beta-cells express hepcidin, an iron-uptake regulatory peptide.

Body iron is involved in various vital functions. Its uptake in the intestine is regulated by hepcidin, a bioactive peptide originally identified in plasma and urine and subsequently in the liver. In the present study, we provide evidence at the transcriptional and translational levels that hepcidin is also expressed in the pancreas of rat and man. Immunohistochemical studies localized the peptide exclusively to beta-cells of the islets of Langerhans. Immunoelectron microscopical analyses revealed that hepcidin is confined to the insulin-storing beta-cell secretory granules. As demonstrated in insulinoma-derived RINm5F cells, the expression of hepcidin in beta-cells is regulated by iron. Based on the present findings we conclude that pancreatic islets are an additional source of the peptide hepcidin. The localization of this peptide to beta-cells suggests that pancreatic beta-cells may be involved in iron metabolism in addition to their genuine function in blood glucose regulation. In view of the various linked iron/glucose disorders in the pancreas, the present findings may provide an insight into the phenomenology of intriguing mutual relationships between iron and glucose metabolisms.

Secretory granules of hypophyseal and pancreatic endocrine cells contain proteins of the neuronal postsynaptic density.

The PDZ domain-containing protein Shank is a master scaffolding protein of the neuronal postsynaptic density and directly or indirectly links neurotransmitter receptors and cell adhesion molecules to the actin-based cytoskeleton. ProSAP/Shank proteins have recently also been detected in several non-neuronal cells in which they are mostly concentrated in the apical subplasmalemmal cytoplasm. In contrast, we have previously reported a more widespread cytoplasmic immunostaining pattern for the ProSAP1/Shank2 protein in endocrine cells at the light-microscopic level. Therefore, in the present study, we have determined the ultrastructural localization of ProSAP1/Shank2 and the ProSAP/Shank-interacting proteins ProSAPiP1 and IRSp53 in pancreatic islet and adenohypophyseal cells by using immunogold staining techniques. Dense immunolabeling of secretory granules including the granule core in cells such as hypophyseal somatotrophs and pancreatic B-cells indicates the unexpected presence of ProSAP/Shank and ProSAP/Shank-interacting proteins in the hormone-storing compartment of endocrine cells. Thus, ProSAP/Shank and certain ProSAP/Shank-interacting proteins exhibit distinct subcellular localizations in the different cell types, raising the possibility that the function of ProSAP/Shank proteins is more diverse than has been envisaged to date.

Expression of postsynaptic density proteins of the ProSAP/Shank family in the thymus.

PSD95-DLG-ZO1 domain-containing proteins of the ProSAP/Shank family are major scaffolding proteins of the neuronal postsynaptic density which play a pivotal role in the linkage of membrane receptors to downstream signal effectors and the actin-based cytoskeleton. Recently, ProSAP1/Shank2 has also been localized in various non-neuronal cells where it may fulfill similar functions as in neurons. We now complement these data by the study of ProSAP/Shank expression at the mRNA and protein level in a primary lymphoid organ, i.e., the thymus. Transcripts for ProSAP1/Shank2, the spliceoform Shank2E, and ProSAP2/Shank3 could be clearly detected in the thymus. Western blot and immunocytochemical analyses verified the presence of ProSAP1/Shank2 and ProSAP2/Shank3 proteins in thymic tissue. Immunoreactivity was concentrated in the whole peripheral cytoplasm of thymocytes underneath the plasma membrane. Discrete subplasmalemmal areas of pronounced ProSAP/Shank immunoreactivity could be demonstrated inside several thymocytes by confocal laser scanning microscopy. Our results establish ProSAP/Shank as a constituent of the cell cortex of thymocytes and thus lead to the hypothesis that ProSAP/Shank proteins serve as a platform for the coordination of membrane receptor-dependent signal transduction in immune cells.

The clathrin interacting protein Clint/epsinR in rat testicular germ cells.

The plasma membrane and the trans-Golgi network (TGN) are major intracellular sites for clathrin-mediated membrane budding. Only recently has the clathrin interacting protein Clint/epsinR/enthoprotin been identified, which is thought to be involved in clathrin-dependent membrane budding from the TGN. Using immunocytochemistry, we now report the presence of Clint in the Golgi region of spermatocytes and spermatids of the rat testis. Together with subcellular fractionation experiments, our data show that, in male germ cells, Clint behaves as a peripheral membrane protein that is probably involved in TGN-related vesicle budding. Moreover, the immunostaining of the acrosome in round and elongating spermatids indicates that Clint operates in membrane traffic between the TGN and the acrosome. It may thus be speculated that the protein is involved in the biogenesis and shaping of acrosomal membranes.

Association of Caldendrin splice isoforms with secretory vesicles in neurohypophyseal axons and the pituitary.

Caldendrin is a neuronal calcium-binding protein, which is highly enriched in the postsynaptic density fraction and exhibits a prominent somato-dendritic distribution in brain. Two additional splice variants derive from the caldendrin gene, which have unrelated N-termini and were previously only detected in the retina. We now show that these isoforms are present in neurohypophyseal axons and on secretory granules of endocrine cells. In light of the described interaction of the Caldendrin C-terminus with Q-type Ca(v)2.1 calcium channels these data suggest that this interaction takes place in neurohypophyseal axons and pituitary cells indicating functions of the short splice variants in triggering Ca2+ transients to a vesicular target interaction.

Brain synaptic junctional proteins at the acrosome of rat testicular germ cells.

Proteins of the presynaptic exocytic machinery have been found associated with the acrosome of male germ cells, suggesting that the sperm acrosome reaction and neurotransmission at chemical synapses may share some common mechanisms. To substantiate this hypothesis, we studied the expression and ultrastructural localization of prominent pre- and postsynaptic protein components in rat testis. The presynaptic membrane trafficking proteins SV2 and complexin, the vesicular amino acid transporters VGLUT and VIAAT, the postsynaptic scaffolding protein ProSAP/Shank, and the postsynaptic calcium-sensor protein caldendrin, could be identified in germ line cells. Immunogold electron microscopy revealed an association of these proteins with the acrosome. In addition, evidence was obtained for the expression of the plasmalemmal glutamate transporters GLT1 and GLAST in rat sperm. The novel finding that not only presynaptic proteins, which are believed to be involved in membrane fusion processes, but also postsynaptic elements are present at the acrosome sheds new light on its structural organization. Moreover, our data point to a possible role for neuroactive amino acids in reproductive physiology.