Regulation of rat Liver Glucokinase Gene Expression by Sterol Regulatory Element Binding Protein-1a and Forkhead box classO1 Transcription factors

Die Glucokinase (GK), auch Hexokinase-IV, katalysiert die Phosphorylierung von Glucose zu Glucose-6-phosphat. Im Gengensatz zu anderen Hexokinasen hat die GK eine geringe Affinität für Glucose, wird durch das Reaktionsprodukt nicht gehemmt und weist eine sigmoidale Kinetik auf, obwohl sie als Monomer vorliegt. Defekte im GK-Gen führen zum maturity-onset diabetes of the young type-2 (Insulin-unabhängig [MODY-2]). So spielt die GK eine wichtige Rolle bei der Aufrechterhaltung der Glucose-Homeostase. Die GK wird vorwiegend in Leber-Hepatozyten, β-Zellen des Pankreas und in einigen neuroendokrinen Zellen des Gastrolintestinaltrakts und des Gehirns exprimiert. In Hepatozyten sind hauptsächlich Insulin und Glucagon für die Regulation der GK-Expression verantwortlich. Hierbei agiert Insulin vorwiegend über den PI3K/PKB-signalweg und moduliert die Aktivität verschiedener Transkriptionsfaktoren, wie z.B. dem Sterol regulatory element binding protein-1 (SREBP-1) und den FoxO/forkhead Transkriptionsfaktoren. Neuere Veröffentlichungen deuten darauf hin, dass sowohl SREBP-1 als auch FoxO1 in antagonistischer Weise die GK-Expression und somit den Glucose-/Lipid-Metabolismus in der Leber beeinflussen. Die genauen Details der SREBP-1 und FoxO1-regulierten GK-Genexpression sind allerdings noch nicht bekannt. Das Ziel dieser Arbeit war daher die Untersuchung der Insulin-abhängigen SREBP-1 und FoxO1- vermittelten Expression des GK-Gens in primären Rattenhepatozyten und in den Hepatomazellen HepG2. Die Stimulation von primaren Rattenhepatozyten mit Insulin induzierte die Expression von GK und SREBP-1. Auch konnte SREBP-1 durch eine Behandlung mit dem LXR-Agonisten TO-901317 induziert werden, was wiederum zu einer Induktion der GK-mRNA Spiegel führte. Computer-Analysen des Leber-spezifischen GK-Promotors zeigten 3 putative SREBP-1 Bindungsstellen (SREs) auf. Transfektionsversuche mit Luciferasegen-Konstrukten, welche durch seriell deletierte GK-Promotoren reguliert werden, deuteten darauf hin, dass eine Sequenz, bekannt als Footprint-B-Seite, in die SREBP-1-abhängige Regulation involviert ist. Weitere Analysen der Footprint-B-Seite, welche in Abschnitt 1 und Abschnitt 2 unterteilt werden kann, zeigten, dass vor allem Abschnitt 2 und weniger Abschnitt 1 für den SREBP-1 Effekt notwendig ist. Des Weiteren konnten durch Mutationsanalysen des GK-Promotors zwei andere Sequenzen, SRE2 und SRE3, identifiziert werden. Transfektionsversuche in primären Hepatozyten und in HepG2-Zellen implizieren, dass diese Elemente Zell-spezifisch verwendet werden. Während in primären Hepatozyten sowohl SRE2 als auch SRE3 für die SREBP-1 vermittelte GK-Promotoraktivität wichtig sind, trägt in HepG2-Zellen nur SRE2 zum SREBP-1-Effekt bei. Zudem ging die SREBP-1-vermittelte Aktivierung des GK-Promotors durch Mutation des HNF-4-Bindungselements verloren, was darauf hindeutet, dass für die vollständige Induktion der GK-Genexpression durch SREBP-1 eine Interaktion dieser Transkriptionsfaktoren notwendig ist. Insulin hat einen dynamischen Effekt auf die FoxO-Transkriptionsfaktoren, welche durch eine PKB-abhängige Phosphorylierung in eine inaktive Form überführt werden und so aus dem Kern ausgeschlossen werden. Hepatozyten, welche mit FoxO1-Expressionsvektoren transfiziert wurden, regulierten ihre GK-mRNA und GK-Promotoraktivität herab. Diese Repression ging durch Stimulation der Hepatozyten mit Insulin verloren. Bekamen Ratten für 48h kein Futter, so war das GK-Protein kaum detektierbar, allerdings war der FoxO1 Proteinspiegel erhöht. Es ist bekannt, dass die transkriptionelle Aktivität der FoxO-Proteine neben Insulin auch durch NAD+ abhängige SIRT1 Deacetylierung reguliert wird. Resveratrol reguliert die mRNA- und Proteinspiegel der GK herab und kehrt den induzierenden Effekt von Insulin um. Ähnliche Resultate wurden für die GK-Enzymaktivität beobachtet. Computer-Analysen des GK-Promotors prognostizierten zwei FoxO1-Bindungselemente (FBEa und FBEb). Überexpression von FoxO1 vermindert die GK-Promotoraktivität in primaren Hepatozyten und in HepG2-Zellen. Mutationen im FoxO1-Bindungselement FBEb beseitigen die FoxO1 vermittelte Repression des GK-Promotors. Des Weiteren führt die Behandlung von Hepatozyten mit dem SIRT1-Aktivator Resveratrol zur Deacetylierung und Aktivierung von FoxO1. Interessanter Weise ging der FoxO1-Effekt auch verloren, wenn die HNF4-Bindungsstelle mutiert war. Dies suggerierte, das FoxO1 mit HNF-4 interagiert und dessen Aktivität reguliert. Insgesamt zeigt die Studie, dass SREBP-1 die GK-Genexpression über die Footprint-B2-Seite, SRE2 und über Interaktion mit HNF-4 aktivieren kann. Obwohl die Interaktion mit HNF-4 auch für die FoxO1-abhängige Regulation des GK- Promotor wichtig ist, konnten zwei zusätzliche Bindungsstellen identifiziert werden. Zum ersten Mal konnte gezeigt weden, dass die FoxO1-Aktivität durch Resveratrol und SIRT1 reguliert wird und dass die Resveratrol-vermittelte Reduktion der GK-Expression auf einer Bindung von FoxO1 am Bindungselement oder auf einer Interaktion mit HNF-4 beruht

Free Fatty Acids in Bone Pathophysiology of Rheumatic Diseases

Obesity—in which free fatty acid (FFA) levels are chronically elevated—is a known risk factor for different rheumatic diseases, and obese patients are more likely to develop osteoarthritis (OA) also in non-weight-bearing joints. These findings suggest that FFA may also play a role in inflammation-related joint damage and bone loss in rheumatoid arthritis (RA) and OA. Therefore, the objective of this study was to analyze if and how FFA influence cells of bone metabolism in rheumatic diseases. When stimulated with FFA, osteoblasts from RA and OA patients secreted higher amounts of the proinflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, growth-related oncogene α, and monocyte chemotactic protein 1. Receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin, and osteoblast differentiation markers were not influenced by FFA. Mineralization activity of osteoblasts correlated inversely with the level of FFA-induced IL-6 secretion. Expression of the Wnt signaling molecules, axin-2 and β-catenin, was not changed by palmitic acid (PA) or linoleic acid (LA), suggesting no involvement of the Wnt signaling pathway in FFA signaling for osteoblasts. On the other hand, Toll-like receptor 4 blockade significantly reduced PA-induced IL-8 secretion by osteoblasts, while blocking Toll-like receptor 2 had no effect. In osteoclasts, IL-8 secretion was enhanced by PA and LA particularly at the earliest time point of differentiation. Differences were observed between the responses of RA and OA osteoclasts. FFA might therefore represent a new molecular factor by which adipose tissue contributes to subchondral bone damage in RA and OA. In this context, their mechanisms of action appear to be dependent on inflammation and innate immune system rather than Wnt-RANKL pathways

Mitochondrial damage by α-synuclein causes cell death in human dopaminergic neurons

Evolving concepts on Parkinson's disease (PD) pathology suggest that α-synuclein (aSYN) promote dopaminergic neuron dysfunction and death through accumulating in the mitochondria. However, the consequence of mitochondrial aSYN localisation on mitochondrial structure and bioenergetic functions in neuronal cells are poorly understood. Therefore, we investigated deleterious effects of mitochondria-targeted aSYN in differentiated human dopaminergic neurons in comparison with wild-type (WT) aSYN overexpression and corresponding EGFP (enhanced green fluorescent protein)-expressing controls. Mitochondria-targeted aSYN enhanced mitochondrial reactive oxygen species (ROS) formation, reduced ATP levels and showed severely disrupted structure and function of the dendritic neural network, preceding neuronal death. Transmission electron microscopy illustrated distorted cristae and many fragmented mitochondria in response to WT-aSYN overexpression, and a complete loss of cristae structure and massively swollen mitochondria in neurons expressing mitochondria-targeted aSYN. Further, the analysis of mitochondrial bioenergetics in differentiated dopaminergic neurons, expressing WT or mitochondria-targeted aSYN, elicited a pronounced impairment of mitochondrial respiration. In a pharmacological compound screening, we found that the pan-caspase inhibitors QVD and zVAD-FMK, and a specific caspase-1 inhibitor significantly prevented aSYN-induced cell death. In addition, the caspase inhibitor QVD preserved mitochondrial function and neuronal network activity in the human dopaminergic neurons overexpressing aSYN. Overall, our findings indicated therapeutic effects by caspase-1 inhibition despite aSYN-mediated alterations in mitochondrial morphology and function

Downregulation of the psychiatric susceptibility gene Cacna1c promotes mitochondrial resilience to oxidative stress in neuronal cells

Affective disorders such as major depression and bipolar disorder are among the most prevalent forms of mental illness and their etiologies involve complex interactions between genetic and environmental risk factors. Over the past ten years, several genome wide association studies (GWAS) have identified CACNA1C as one of the strongest genetic risk factors for the development of affective disorders. However, its role in disease pathogenesis is still largely unknown. Vulnerability to affective disorders also involves diverse environmental risk factors such as perinatal insults, childhood maltreatment, and other adverse pathophysiological or psychosocial life events. At the cellular level, such environmental influences may activate oxidative stress pathways, thereby altering neuronal plasticity and function. Mitochondria are the key organelles of energy metabolism and, further, highly important for the adaptation to oxidative stress. Accordingly, multiple lines of evidence including post-mortem brain and neuro-imaging studies suggest that psychiatric disorders are accompanied by mitochondrial dysfunction. In this study, we investigated the effects of Cacna1c downregulation in combination with glutamate-induced oxidative stress on mitochondrial function, Ca2+ homeostasis, and cell viability in mouse hippocampal HT22 cells. We found that the siRNA-mediated knockdown of Cacna1c preserved mitochondrial morphology, mitochondrial membrane potential, and ATP levels after glutamate treatment. Further, Cacna1c silencing inhibited excessive mitochondrial reactive oxygen species formation and calcium influx, and protected the HT22 cells from oxidative cell death. Overall, our findings suggest that the GWAS-confirmed psychiatric risk gene CACNA1C plays a major role in oxidative stress pathways with particular impact on mitochondrial integrity and function.ISSN:2058-771

FoxO1 and HNF-4 are involved in regulation of hepatic glucokinase gene expression by resveratrol

Abstract Resveratrol, a polyphenol derived from grapes, exerts important effects on glucose and lipid metabolism, yet detailed mechanisms mediating these effects remain unknown. The liver plays a central role in energy homeostasis, and glucokinase (GK) is a key enzyme involved in glucose utilization. Resveratrol activates SIRT1 (sirtuin 1), which promotes deacetylation of the forkhead transcription factor FoxO1. Previously, we reported that FoxO1 can suppress and that HNF-4 can stimulate GK expression in the liver. Here, we examined the role of FoxO1 and HNF-4 in mediating resveratrol effects on liver GK expression. Resveratrol suppressed hepatic GK expression in vivo and in isolated hepatocytes, and knocking down FoxO1 with shRNAs disrupted this effect. Reporter gene, gel shift, supershift assay, and chromatin immunoprecipitation studies show that FoxO1 binds to the GK promoter and that the interplay between FoxO1 and HNF-4 within the GK promoter is essential for mediating the effects of resveratrol. Resveratrol promotes deacetylation of FoxO1 and enhances its recruitment to the FoxO-binding element. Conversely, resveratrol suppresses recruitment of HNF-4 to its binding site, and knockdown of FoxO1 blocks this effect of resveratrol. Coprecipitation and chromatin immunoprecipitation studies show that resveratrol enhances interaction between FoxO1 and HNF-4, reduces binding of HNF-4 to its own site, and promotes its recruitment to the FoxO site in a FoxO1-dependent manner. These results provide the first evidence that resveratrol represses GK expression via FoxO1 and that the interaction between FoxO1 and HNF-4 contributes to these effects of resveratrol

BID links ferroptosis to mitochondrial cell death pathways

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the Xc(-) system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by Xc(-) inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death