Genomveränderungen–CRISPR/Cas9 als Methode der Wahl oder Qual?

Abstract The last years showed the rise of CRISPR/Cas9 to stardom. Several features such as programmability, specificity, and efficiency, make it to be a technique of choice for genome editing even in processes where basic aspects are not understood. However, scientific, technical and society driven challenges remain. To cope with these challenges, further research, technological improvements, translational studies as well as an open exchange between laboratories and the society are required

Hypoxia-inducible factors (HIFs) and phosphorylation:impact on stability, localization, and transactivity

Abstract The hypoxia-inducible factor α-subunits (HIFα) are key transcription factors in the mammalian response to oxygen deficiency. The HIFα regulation in response to hypoxia occurs primarily on the level of protein stability due to posttranslational hydroxylation and proteasomal degradation. However, HIF α-subunits also respond to various growth factors, hormones, or cytokines under normoxia indicating involvement of different kinase pathways in their regulation. Because these proteins participate in angiogenesis, glycolysis, programmed cell death, cancer, and ischemia, HIFα regulating kinases are attractive therapeutic targets. Although numerous kinases were reported to regulate HIFα indirectly, direct phosphorylation of HIFα affects HIFα stability, nuclear localization, and transactivity. Herein, we review the role of phosphorylation-dependent HIFα regulation with emphasis on protein stability, subcellular localization, and transactivation

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

Cyclin-dependent kinase 5 (CDK5)-mediated phosphorylation of upstream stimulatory factor 2 (USF2) contributes to carcinogenesis

Abstract The transcription factor USF2 is supposed to have an important role in tumor development. However, the regulatory mechanisms contributing to the function of USF2 are largely unknown. Cyclin-dependent kinase 5 (CDK5) seems to be of importance since high levels of CDK5 were found in different cancers associated with high USF2 expression. Here, we identified USF2 as a phosphorylation target of CDK5. USF2 is phosphorylated by CDK5 at two serine residues, serine 155 and serine 222. Further, phosphorylation of USF2 at these residues was shown to stabilize the protein and to regulate cellular growth and migration. Altogether, these results delineate the importance of the CDK5-USF2 interplay in cancer cells

Hypoxia ameliorates maternal diet-induced insulin resistance during pregnancy while having a detrimental effect on the placenta

Abstract Maternal overweight/obesity contributes significantly to the development of gestational diabetes, which causes risks to both mother and fetus and is increasing sharply in prevalence worldwide. Since hypoxia reprograms energy metabolism and can alleviate weight gain, adiposity, insulin resistance (IR), and dyslipidemia, we set out to study the potential of sustained reduced ambient oxygen tension (15% O2) during pregnancy for alleviating the detrimental effects of diet-induced IR in C57Bl/6N mice, taking normal chow-fed and normoxia (21% O2) groups as controls. Our data show that hypoxic intervention reduced maternal weight gain, adiposity, and adipose tissue inflammation, and ameliorated maternal glucose metabolism and IR during gestation in diet-induced IR relative to normoxia. Where diet-induced IR reduced maternal hemoglobin and increased serum erythropoietin levels, hypoxic intervention compensated for these changes. Diet-induced IR reduced fetal growth in normoxia, and even more in hypoxia. Hypoxic intervention reduced liver weight gain during pregnancy in the dams with diet-induced IR, maternal liver weight being positively associated with embryo number. In case of diet-induced IR, the hypoxic intervention compromised placental energy metabolism and vascularization and increased end-pregnancy placental necrosis. Altogether, these data show that although hypoxic intervention mediates several beneficial effects on maternal metabolism, the combination of it with diet-induced IR is even more detrimental to the placental and fetal outcome than diet-induced IR alone

Hypoxia causes reductions in birth weight by altering maternal glucose and lipid metabolism

Abstract Hypoxia of residence at high altitude (>2500 m) decreases birth weight. Lower birth weight associates with infant mortality and morbidity and increased susceptibility to later-in-life cardiovascular and metabolic diseases. We sought to determine the effects of hypoxia on maternal glucose and lipid metabolism and their contributions to fetal weight. C57BL6/NCrl mice, housed throughout gestation in normobaric hypoxia (15% oxygen) or normoxia, were studied at mid (E9.5) or late gestation (E17.5). Fetal weight at E17.5 was 7% lower under hypoxia than normoxia. The hypoxic compared with normoxic dams had ~20% less gonadal white adipose tissue at mid and late gestation. The hypoxic dams had better glucose tolerance and insulin sensitivity compared with normoxic dams and failed to develop insulin resistance in late gestation. They also had increased glucagon levels. Glucose uptake to most maternal tissues was ~2-fold greater in the hypoxic than normoxic dams. The alterations in maternal metabolism in hypoxia were associated with upregulation of hypoxia-inducible factor (HIF) target genes that serve, in turn, to increase glycolytic metabolism. We conclude that environmental hypoxia alters maternal metabolism by upregulating the HIF-pathway, and suggest that interventions that antagonize such changes in metabolism in high-altitude pregnancy may be helpful for preserving fetal growth

Resveratrol suppresses PAI-1 gene expression in a human in vitro model of inflamed adipose tissue

Abstract Increased plasminogen activator inhibitor-1 (PAI-1) levels are associated with a number of pathophysiological complications; among them is obesity. Resveratrol was proposed to improve obesity-related health problems, but the effect of resveratrol on PAI-1 gene expression in obesity is not completely understood. In this study, we used SGBS adipocytes and a model of human adipose tissue inflammation to examine the effects of resveratrol on the production of PAI-1. Treatment of SGBS adipocytes with resveratrol reduced PAI-1 mRNA and protein in a time- and concentration-dependent manner. Further experiments showed that obesity-associated inflammatory conditions lead to the upregulation of PAI-1 gene expression which was antagonized by resveratrol. Although signaling via PI3K, Sirt1, AMPK, ROS, and Nrf2 appeared to play a significant role in the modulation of PAI-1 gene expression under noninflammatory conditions, those signaling components were not involved in mediating the resveratrol effects on PAI-1 production under inflammatory conditions. Instead, we demonstrate that the resveratrol effects on PAI-1 induction under inflammatory conditions were mediated via inhibition of the NFκB pathway. Together, resveratrol can act as NFκB inhibitor in adipocytes and thus the subsequently reduced PAI-1 expression in inflamed adipose tissue might provide a new insight towards novel treatment options of obesity

Obesity and inflammation:reduced cytokine expression due to resveratrol in a human in vitro model of inflamed adipose tissue

Abstract Obesity is associated with an inflammatory status and linked with a number of pathophysiological complications among them cardiovascular disease, type 2 diabetes mellitus, or the metabolic syndrome. Resveratrol was proposed to improve obesity-related inflammatory problems, but the effect of resveratrol on cytokine expression in obesity is not completely understood. In this study, we used an in vitro model of human adipose tissue inflammation to examine the effects of resveratrol on the production of the inflammatory cytokines interleukin 6 (IL-6), IL-8, and monocyte chemoattractant protein 1 (MCP-1). We found that resveratrol reduced IL-6, IL-8, and MCP-1 levels in a concentration-dependent manner in adipocytes under inflammatory conditions. Further experiments showed that the action of resveratrol was mainly due to its NFκB inhibitory potential. Thus, our data support the concept that resveratrol can alleviate obesity-induced up-regulation of inflammatory cytokines providing a new insight toward novel treatment options in obesity

Human phytanoyl-CoA dioxygenase domain-containing 1 (PHYHD1) is a putative oxygen sensor associated with RNA and carbohydrate metabolism

Abstract Human phytanoyl-CoA dioxygenase domain-containing 1 (PHYHD1) is a 2-oxoglutarate (2OG)-dependent dioxygenase implicated in Alzheimer’s disease, some cancers, and immune cell functions. The substrate, kinetic and inhibitory properties, function and subcellular localization of PHYHD1 are unknown. We used recombinant expression and enzymatic, biochemical, biophysical, cellular and microscopic assays for their determination. The apparent Km values of PHYHD1 for 2OG, Fe²⁺ and O₂ were 27, 6 and > 200 μm, respectively. PHYHD1 activity was tested in the presence of 2OG analogues, and it was found to be inhibited by succinate and fumarate but not R-2-hydroxyglutarate, whereas citrate acted as an allosteric activator. PHYHD1 bound mRNA, but its catalytic activity was inhibited upon interaction. PHYHD1 was found both in the nucleus and cytoplasm. Interactome analyses linked PHYHD1 to cell division and RNA metabolism, while phenotype analyses linked it to carbohydrate metabolism. Thus, PHYHD1 is a potential novel oxygen sensor regulated by mRNA and citrate

Genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) reduces atherosclerotic plaques in mice

Abstract Objective: Atherosclerosis is a key component of cardiovascular diseases. We set out to study here whether genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) could protect against atherosclerosis as does inhibition of the other 3 classical HIF-P4Hs (hypoxia-inducible factor prolyl 4-hydroxylases). Approach and Results: We generated a double knockout mouse line deficient in P4H-TM and LDL (low-density lipoprotein) receptor (P4h-tm−/−/Ldlr−/−) and subjected these mice to a high-fat diet for 13 weeks. The double knockout mice had less atherosclerotic plaques in their full-length aorta than their P4h-tm+/+/Ldlr−/− counterparts and also had lower serum triglyceride levels on standard laboratory diet and high-fat diet, higher levels of IgM autoantibodies against Ox-LDL (oxidized LDL), and significantly higher lipoprotein lipase protein levels in white adipose tissue and sera. RNA-sequencing analysis revealed changes in expression of mRNAs in multiple pathways including lipid metabolism and immunologic response in the P4h-tm−/−/Ldlr−/− livers as compared with P4h-tm+/+/Ldlr−/−. Conclusions: Our data identify P4H-TM inhibition as a potential novel immuno-metabolic mechanism for intervening in the pathology of atherosclerosis, as hypertriglyceridemia is an individual risk factor for atherosclerosis, and IgM antibodies to Ox-LDL and increased lipoprotein lipase have been associated with protection against it