ABSTRACT
AIMS/HYPOTHESIS: Prolonged exposure of beta cells to low glucose concentrations triggers their apoptosis and is known to activate AMP-activated protein kinase (AMPK) in beta cell lines. We examined whether prolonged activation of AMPK can trigger apoptosis in rodent beta cells. METHODS: Primary beta cells were FACS-purified from rats, and from wild-type and AMPK(alpha2)-deficient mice. AMPK activation in beta cells was induced by the adenosine analog AICA-riboside and detected by immunoblotting using a phosphospecific antibody. Apoptosis of rodent beta cells was monitored by FACS analysis of beta cell DNA content, by direct counting of apoptotic cells using fluorescence microscopy, or by measurement of their caspase-3 activity. RESULTS: Dose-dependent and time-dependent apoptosis of the cells, concommittant with an activation of caspase-3, were suppressed by the caspase inhibitors zVAD-fmk and zDEVD-fmk. Apoptosis induction by AICA-riboside was also prevented by adding the MAPK-inhibitor SB203580 which blocked the AICA-riboside-induced phosphorylation of AMPK. Beta cells isolated from AMPK-(alpha2)-deficient mice were resistant against AICA-riboside induced apoptosis. CONCLUSION/INTERPRETATION: Sustained activation of AMPK by AICA-riboside can trigger a caspase-dependent apoptosis of pancreatic beta cells.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Apoptosis , Islets of Langerhans/drug effects , Islets of Langerhans/physiology , Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , Ribonucleosides/pharmacology , AMP-Activated Protein Kinases , Animals , Caspase 3 , Caspases/metabolism , Cells, Cultured , Enzyme Activation , Mice , Multienzyme Complexes/deficiency , Protein Serine-Threonine Kinases/deficiency , RatsABSTRACT
The discovery of the AMP-activated protein kinase (AMPK) more than a decade ago has shed much light on the cellular response to stresses characterized by a fall in the concentration of ATP and an increase in the AMP/ATP ratio. All conditions known to increase this ratio activate AMPK, whose major role is to act as an emergency signal to conserve ATP. It does so by inhibiting anabolic processes and by activating pathways producing ATP. In recent years, our laboratory has discovered new targets of AMPK. The purpose of this short review is to summarize our contribution to this field.
Subject(s)
Apoptosis , Mitogen-Activated Protein Kinases/metabolism , Multienzyme Complexes/metabolism , Multienzyme Complexes/physiology , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , AMP-Activated Protein Kinases , Adenosine Triphosphate/metabolism , Animals , Humans , JNK Mitogen-Activated Protein Kinases , Liver/metabolism , Phosphorylation , Protein Structure, TertiaryABSTRACT
In this paper, we demonstrated that in cultured rat hepatocytes cell swelling induced the activation of STAT1 and STAT3 proteins without any effect on STAT4, STAT5 and STAT6 proteins. Cell swelling induced an activation of STAT proteins through an increase in the phosphorylation of the tyrosine residue also phosphorylated by interleukin-6, but without any activation of JAK kinases. The signaling pathway by which cell swelling activated STAT1 and STAT3 is discussed.
Subject(s)
DNA-Binding Proteins/metabolism , Liver/metabolism , Trans-Activators/metabolism , Animals , Cell Size , Cells, Cultured , Gene Expression Regulation , Interleukin-6/pharmacology , Janus Kinase 3 , Liver/cytology , Male , Osmolar Concentration , Phosphorylation/drug effects , Protein-Tyrosine Kinases/analysis , Rats , Rats, Wistar , STAT1 Transcription Factor , STAT2 Transcription FactorABSTRACT
The effect of cell swelling on the expression of the alpha2-macroglobulin (alpha2M) gene was studied in hepatocytes in culture. Hypoosmolarity induced an increase (3-fold increase) in the level of alpha2M mRNA through a corresponding stimulation of the rate of transcription of the alpha2M gene. The addition of raffinose (100 mM) corrected the effect of hypoosmolarity at both mRNA and transcriptional level, demonstrating that cell swelling per se was responsible for the observed effect on the expression of the alpha2M gene. Moreover, the effect of cell swelling was additive to that of interleukin 6, a major mediator of the acute-phase response.
Subject(s)
Alpha-Globulins/genetics , Cell Size/physiology , Gene Expression Regulation , Liver/metabolism , Alpha-Globulins/biosynthesis , Animals , Cells, Cultured , Interleukin-6/physiology , Liver/cytology , Male , RNA, Messenger/metabolism , Rats , Rats, Wistar , Transcription, GeneticABSTRACT
Glutamine is able to regulate the expression of various genes in rat hepatocytes. This includes genes coding for proteins involved in glutamine utilization, such as argininosuccinate synthetase (ureagenesis) or phosphoenolpyruvate carboxykinase (gluconeogenesis). Moreover, glutamine is also able to stimulate the expression of genes involved in the acute-phase response, such as the alpha 2-macroglobulin gene. The effect of glutamine on the regulation of gene expression may be explained, at least in part, by the cell swelling due to its sodium-dependent transport. The physiological significance of the effect of glutamine is discussed.
Subject(s)
Gene Expression Regulation , Glutamine/physiology , Liver/metabolism , Aminoisobutyric Acids/metabolism , Animals , Argininosuccinate Synthase/genetics , Argininosuccinate Synthase/metabolism , Cell Size/physiology , Dose-Response Relationship, Drug , Interleukin-6/metabolism , Models, Biological , Phosphoenolpyruvate Carboxykinase (ATP)/genetics , Phosphoenolpyruvate Carboxykinase (ATP)/metabolism , Rats , Time Factors , alpha-Macroglobulins/genetics , alpha-Macroglobulins/metabolismABSTRACT
The repertoire of the actions of specific amino acids on gene expression is relatively limited in mammalian cells. Glutamine constitutes the most studied amino acid and recent works intended to demonstrate its mechanism of action on two genes: the beta-actin and the phosphoenolpyruvate carboxykinase genes. From these studies, it appears that glutamine may regulate gene expression by, at least, two different mechanisms: one through the glutamine-induced cell swelling, and another through its intracellular metabolism. The involvement of phosphatidylinositol 3-kinase in the signaling pathway triggered by cell swelling is discussed.
