ABSTRACT
AIMS/HYPOTHESIS: Using primary cultures of human pancreatic islets, purified human pancreatic beta cells and the mouse beta TC6-F7 cell line, we analysed the expression of nerve growth factor, (NGF/NGF) receptors in beta cells. To investigate whether NGF could sub-serve an autocrine antiapoptotic role in beta cells, we studied the effects of NGF withdrawal using a neutralizing monoclonal anti-NGF antibody. METHODS: The expression of NGF and NGF receptors (gp140(Trk-A) and p75(NTR)) were analysed by RT-PCR and immunofluorescence. Pulse-chase experiments and beta cell/PC12 co-cultures were used to investigate NGF production and secretion from beta cells. Possible apoptosis induced by NGF withdrawal was monitored by phosphatidylserine translocation, nucleosomal formation, DNA laddering and FACS analysis. Involvement of transcription/translation mechanisms were investigated as well as the gp140(Trk-A) required. Finally, signal transduction pathways typically involved in apoptotic mechanisms were analysed by western blot analysis. RESULTS: We show that NGF and both NGF receptors, gp140(Trk-A) and p75(NTR) are expressed in beta cells where NGF is produced and secreted in a biologically active form. NGF-withdrawal induces beta-cell transcription/translation independent apoptosis but mediated by gp140(Trk-A). Analysis of signal transduction pathways revealed that NGF withdrawal inhibits the PI3-K, protein kinase B (AKT), Bad survival pathway and activates c-Jun kinase (JNK) whereas ERKs and p38 mitogen-activated protein kinase (MAPK) are not affected. Moreover, Bcl-XL, but not Bcl-2 protein expression are reduced. CONCLUSION/INTERPRETATION: We suggest that the integrity of the NGF/NGF receptor system and NGF bioavailability participate in controlling beta-cell survival in culture which represents a key issue for improving possibilities for transplantations in the treatment of diabetes.
Subject(s)
Apoptosis , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , JNK Mitogen-Activated Protein Kinases , Nerve Growth Factor/physiology , Protein Serine-Threonine Kinases , Animals , Antibodies, Monoclonal/pharmacology , Carrier Proteins/metabolism , Cell Line , Cells, Cultured , Coculture Techniques , DNA Fragmentation , Enzyme Activation , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression , Humans , MAP Kinase Kinase 4 , Male , Mice , Mice, Transgenic , Middle Aged , Mitogen-Activated Protein Kinase Kinases/metabolism , Nerve Growth Factor/genetics , Nerve Growth Factor/immunology , Nucleosomes/ultrastructure , PC12 Cells , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylserines/metabolism , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/analysis , Rats , Receptor, Nerve Growth Factor , Receptor, trkA/analysis , Receptor, trkA/genetics , Receptors, Nerve Growth Factor/analysis , Receptors, Nerve Growth Factor/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , bcl-Associated Death Protein , bcl-X ProteinABSTRACT
CONTEXT: It is worth noting that islets and betaTC6-F7 cells share a common pattern of expression of neurotrophins and neurotrophin receptors. Recently, several studies have hypothesized a role for nerve growth factor in pancreatic development and maturation, suggesting that nerve growth factor may be a survival factor for pancreatic beta-cells. OBJECTIVE: The aim of the present study was to investigate the pattern of expression of neurotrophins and their relative receptors both in rat pancreatic islets and in a wide panel of insulinoma cell lines. MAIN OUTCOME MEASURES: A semi-quantitative reverse-transcription polymerase chain reaction analysis was performed on ribonucleic acids extracted from these cells. RESULTS: Reverse transcription-polymerase chain reaction analysis demonstrates that brain-derived neurotrophic factor, as well as neurotrophins 3 and 4, are expressed both in islets and in all insulinoma cells, while nerve growth factor is expressed only in islets, betaTC6-F7 cells and, at a low level, in RIN 1046-38 cells. Receptors protein tyrosine kinase A and C are ubiquitously expressed both in islets and insulinoma cells. Tyrosine kinase B is absent in HIT-T15 cells. CONCLUSIONS: These data indicate that betaTC6-F7 cells are a suitable model for studying the role of neurotrophins in the survival of beta-cells.
Subject(s)
Insulinoma/metabolism , Islets of Langerhans/chemistry , Islets of Langerhans/metabolism , Nerve Growth Factors/biosynthesis , RNA, Messenger/biosynthesis , Receptors, Nerve Growth Factor/biosynthesis , Animals , Cell Survival/genetics , Cricetinae , Gene Expression Profiling/methods , Insulinoma/genetics , Insulinoma/pathology , Mice , Mice, Transgenic , Nerve Growth Factors/genetics , Rats , Receptors, Nerve Growth Factor/genetics , Tumor Cells, CulturedABSTRACT
Voltage-dependent anion channels (VDACs) are a family of pore-forming proteins encoded by different genes, with at least three protein products expressed in mammalian tissues. The major recognized functional role of VDACs is to permit the almost free permeability of the outer mitochondrial membrane (OMM). Although VDAC1 is the best known among VDAC isoforms, its exclusively mitochondrial location is still debated. Therefore, we have measured its co-localization with markers of cellular organelles or compartments in skeletal muscle fibers by single or double immunofluorescence and traditional as well as confocal microscopy. Our results show that VDAC1 immunoreactivity corresponds to mitochondria and sarcoplasmic reticulum, while sarcolemmal reactivity, previously reported, was not observed. Since VDAC1 has been suggested to be involved in the control of oxidative phosphorylation, we sought for possible gene regulation of VDAC1, VDAC2 and VDAC3 in skeletal muscle of the dystrophin-deficient mdx mouse, which suffers of an impaired control of energy metabolism. Our results show that, while VDAC1 mRNA and protein and VDAC2 mRNA are normally expressed. VDAC3 mRNA is markedly down-regulated in mdx mouse muscle at different ages (before, during and after the outburst of myofiber necrosis). This finding suggests a possible involvement of VDAC3 expression in the early pathogenic events of the mdx muscular dystrophy.
Subject(s)
Cell Compartmentation/physiology , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Muscular Dystrophies/metabolism , Porins/genetics , Protein Isoforms/genetics , Animals , Down-Regulation/genetics , Humans , Mice , Mice, Inbred C57BL/genetics , Mice, Inbred C57BL/metabolism , Mice, Inbred mdx/genetics , Mice, Inbred mdx/metabolism , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Membrane Transport Proteins , Muscle Fibers, Skeletal/ultrastructure , Muscle, Skeletal/ultrastructure , Muscular Dystrophies/genetics , Muscular Dystrophies/physiopathology , RNA, Messenger/metabolism , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum/ultrastructure , Voltage-Dependent Anion Channel 1 , Voltage-Dependent Anion Channel 2 , Voltage-Dependent Anion ChannelsABSTRACT
It has been previously demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) regulates insulin secretion. PACAP exerts its biological action by binding to at least three different receptor subtypes coupled to different signal transduction mechanisms. The signaling pathways underlying the insulinotropic effect of PACAP involve mainly the activation of adenylate cyclase to form cAMP, which directly and indirectly, through increased intracellular Ca2+, stimulates insulin exocytosis. In the present study we have characterized the functional and molecular expression of PACAP/vasoactive intestinal polypeptide receptors isoforms and subtypes and its isoforms in a beta-cell line and in isolated rat pancreatic islets. Although insulinoma cells express the messenger RNA encoding PAC1 (-R and -hop variants), VPAC1 and VPAC2, binding experiments indicate the preponderance of PAC1 over VPAC 1-2 receptors. We have also shown that the main signaling pathway of PACAP in beta-cells is mediated by adenylate cyclase, whereas the inositol 1,4,5-trisphosphate pathway is almost inactive. Furthermore, we have demonstrated that PACAP exerts long-term effects on beta-cells, such as transcriptional regulation of the insulin gene and genes of the glucose-sensing system (GLUT1 and hexokinase 1).
