Markus Heine

Coupling governs entrainment range of circadian clocks.

Circadian clocks are endogenous oscillators driving daily rhythms in physiology and behavior. Synchronization of these timers to environmental light–dark cycles (‘entrainment’) is crucial for an organisms fitness. Little is known about which oscillator qualities determine entrainment, i.e., entrainment range, phase and amplitude. In a systematic theoretical and experimental study, we uncovered these qualities for circadian oscillators in the suprachiasmatic nucleus (SCN—the master clock in mammals) and the lung (a peripheral clock): (i) the ratio between stimulus (zeitgeber) strength and oscillator amplitude and (ii) the rigidity of the oscillatory system (relaxation rate upon perturbation) determine entrainment properties. Coupling among oscillators affects both qualities resulting in increased amplitude and rigidity. These principles explain our experimental findings that lung clocks entrain to extreme zeitgeber cycles, whereas SCN clocks do not. We confirmed our theoretical predictions by showing that pharmacological inhibition of coupling in the SCN leads to larger ranges of entrainment. These differences between master and the peripheral clocks suggest that coupling‐induced rigidity in the SCN filters environmental noise to create a robust circadian system.

Regulation of Clock-Controlled Genes in Mammals.

The complexity of tissue- and day time-specific regulation of thousands of clock-controlled genes (CCGs) suggests that many regulatory mechanisms contribute to the transcriptional output of the circadian clock. We aim to predict these mechanisms using a large scale promoter analysis of CCGs. Our study is based on a meta-analysis of DNA-array data from rodent tissues. We searched in the promoter regions of 2065 CCGs for highly overrepresented transcription factor binding sites. In order to compensate the relatively high GC-content of CCG promoters, a novel background model to avoid a bias towards GC-rich motifs was employed. We found that many of the transcription factors with overrepresented binding sites in CCG promoters exhibit themselves circadian rhythms. Among the predicted factors are known regulators such as CLOCK∶BMAL1, DBP, HLF, E4BP4, CREB, RORα and the recently described regulators HSF1, STAT3, SP1 and HNF-4α. As additional promising candidates of circadian transcriptional regulators PAX-4, C/EBP, EVI-1, IRF, E2F, AP-1, HIF-1 and NF-Y were identified. Moreover, GC-rich motifs (SP1, EGR, ZF5, AP-2, WT1, NRF-1) and AT-rich motifs (MEF-2, HMGIY, HNF-1, OCT-1) are significantly overrepresented in promoter regions of CCGs. Putative tissue-specific binding sites such as HNF-3 for liver, NKX2.5 for heart or Myogenin for skeletal muscle were found. The regulation of the erythropoietin (Epo) gene was analysed, which exhibits many binding sites for circadian regulators. We provide experimental evidence for its circadian regulated expression in the adult murine kidney. Basing on a comprehensive literature search we integrate our predictions into a regulatory network of core clock and clock-controlled genes. Our large scale analysis of the CCG promoters reveals the complexity and extensiveness of the circadian regulation in mammals. Results of this study point to connections of the circadian clock to other functional systems including metabolism, endocrine regulation and pharmacokinetics.

Tailor-Made Quantum Dot and Iron Oxide Based Contrast Agents for in Vitro and in Vivo Tumor Imaging

The biofunctionalization of CdSe/CdS/ZnS quantum dots and Fe(3)O(4) nanocrystals using a novel ligand system based on polyisoprene-block-poly(ethylene oxide) ligands is described. The synthesis includes a partial ligand exchange of the hydrophobic nanocrystals with amino-functionalized polyisoprene ligands, followed by seeded micelle formation of the diblock-copolymers in water. The resulting water-soluble quantum dots showed fluorescence quantum efficiencies in the 40 to 50% range and extraordinary fluorescence stability in the biological environment after cross-linking of the polyisoprene moiety of the ligand shell. No toxicity was detected by water-soluble tetrazolium (WST8) and lactate dehydrogenase (LDH) assays, even at very high nanoparticle concentrations, and almost no nonspecific cell adhesion was detected. The ligand shell was further coupled to the antigen-related cell adhesion molecule (CEACAM) specific monoclonal antibody T84.1. The so-conjugated Fe(3)O(4) nanocrystals allowed in vitro and in vivo tumor targeting by magnetic resonance imaging.

TGF-β-dependent induction of CD4+CD25+Foxp3 + Tregs by liver sinusoidal endothelial cells

BACKGROUND & AIMS CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction. METHODS To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3(-) non-Tregs into Foxp3(+) Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-β was tested in Treg conversion assays using T cells with reduced TGF-β sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE). RESULTS All tested liver cell types were capable of inducing Foxp3(+) Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-β. LSECs featured membrane-bound LAP/TGF-β and the anchor molecule GARP, which is required for tethering LAP/TGF-β to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE. CONCLUSIONS We demonstrate that LSECs are the major liver cell type responsible for TGF-β dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-β to their membrane.

ANGPTL4 mediates shuttling of lipid fuel to brown adipose tissue during sustained cold exposure

Brown adipose tissue (BAT) activation via cold exposure is increasingly scrutinized as a potential approach to ameliorate cardio-metabolic risk. Transition to cold temperatures requires changes in the partitioning of energy substrates, re-routing fatty acids to BAT to fuel non-shivering thermogenesis. However, the mechanisms behind the redistribution of energy substrates to BAT remain largely unknown. Angiopoietin-like 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL) activity, is highly expressed in BAT. Here, we demonstrate that ANGPTL4 is part of a shuttling mechanism that directs fatty acids derived from circulating triglyceride-rich lipoproteins to BAT during cold. Specifically, we show that cold markedly down-regulates ANGPTL4 in BAT, likely via activation of AMPK, enhancing LPL activity and uptake of plasma triglyceride-derived fatty acids. In contrast, cold up-regulates ANGPTL4 in WAT, abolishing a cold-induced increase in LPL activity. Together, our data indicate that ANGPTL4 is an important regulator of plasma lipid partitioning during sustained cold. DOI: http://dx.doi.org/10.7554/eLife.08428.001

A Simple and Widely Applicable Method to 59Fe-Radiolabel Monodisperse Superparamagnetic Iron Oxide Nanoparticles for In Vivo Quantification Studies

A simple, fast, efficient, and widely applicable method to radiolabel the cores of monodisperse superparamagnetic iron oxide nanoparticles (SPIOs) with (59)Fe was developed. These cores can be used as precursors for a variety of functionalized nanodevices. A quality control using filtration techniques, size-exclusion chromatography, chemical degradation methods, transmission electron microscopy, and magnetic resonance imaging showed that the nanoparticles were stably labeled with (59)Fe. Furthermore, the particle structure and the magnetic properties of the SPIOs were unchanged. In a second approach, monodisperse SPIOs stabilized with (14)C-oleic acid were synthesized, and the stability of this shell labeling was studied. In proof of principle experiments, the (59)Fe-SPIOs coated with different shells to make them water-soluble were used to evaluate and compare in vivo pharmacokinetic parameters such as blood half-life. It could also be shown that our radiolabeled SPIOs embedded in recombinant lipoproteins can be used to quantify physiological processes in closer detail than hitherto possible. In vitro and in vivo experiments showed that the (59)Fe label is stable enough to be applied in vivo, whereas the (14)C label is rapidly removed from the iron core and is not adequate for in vivo studies. To obtain meaningful results in in vivo experiments, only (59)Fe-labeled SPIOs should be used.

FGF21 Lowers Plasma Triglycerides by Accelerating Lipoprotein Catabolism in White and Brown Adipose Tissues

FGF21 decreases plasma triglycerides (TGs) in rodents and humans; however, the underlying mechanism or mechanisms are unclear. In the present study, we examined the role of FGF21 in production and disposal of TG-rich lipoproteins (TRLs) in mice. Treatment with pharmacological doses of FGF21 acutely reduced plasma non-esterified fatty acids (NEFAs), liver TG content, and VLDL-TG secretion. In addition, metabolic turnover studies revealed that FGF21 facilitated the catabolism of TRL in white adipose tissue (WAT) and brown adipose tissue (BAT). FGF21-dependent TRL processing was strongly attenuated in CD36-deficient mice and transgenic mice lacking lipoprotein lipase in adipose tissues. Insulin resistance in diet-induced obese and ob/ob mice shifted FGF21 responses from WAT toward energy-combusting BAT. In conclusion, FGF21 lowers plasma TGs through a dual mechanism: first, by reducing NEFA plasma levels and consequently hepatic VLDL lipidation and, second, by increasing CD36 and LPL-dependent TRL disposal in WAT and BAT.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes.

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via “wick-in-needle” technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.

Exceedingly small iron oxide nanoparticles as positive MRI contrast agents

Significance Gadolinium (Gd)-based contrast agents (GBCAs) are currently the mainstream clinical MRI contrast agents. Some GBCAs have shown a long-term toxicity—nephrogenic systemic fibrosis (NSF)—and Gd depositions in the brain. The NSF has triggered a Food and Drug Administration (FDA) black-box warning and a contraindication of some GBCAs. The finding of Gd depositions led to an ongoing FDA investigation to monitor their possible long-term adverse effects. Here, we present T1-weighted contrast-enhanced MR imaging and angiography using zwitterion-coated exceedingly small superparamagnetic iron oxide nanoparticles (ZES-SPIONs) in mice and rats. Renal clearance and biodistribution results further demonstrate that ZES-SPIONs are qualitatively different from previously reported SPIONs. This work may open up opportunities to develop exceedingly small SPIONs that show effective T1 contrast as Gd-free alternatives to GBCAs. Medical imaging is routine in the diagnosis and staging of a wide range of medical conditions. In particular, magnetic resonance imaging (MRI) is critical for visualizing soft tissue and organs, with over 60 million MRI procedures performed each year worldwide. About one-third of these procedures are contrast-enhanced MRI, and gadolinium-based contrast agents (GBCAs) are the mainstream MRI contrast agents used in the clinic. GBCAs have shown efficacy and are safe to use with most patients; however, some GBCAs have a small risk of adverse effects, including nephrogenic systemic fibrosis (NSF), the untreatable condition recently linked to gadolinium (Gd) exposure during MRI with contrast. In addition, Gd deposition in the human brain has been reported following contrast, and this is now under investigation by the US Food and Drug Administration (FDA). To address a perceived need for a Gd-free contrast agent with pharmacokinetic and imaging properties comparable to GBCAs, we have designed and developed zwitterion-coated exceedingly small superparamagnetic iron oxide nanoparticles (ZES-SPIONs) consisting of ∼3-nm inorganic cores and ∼1-nm ultrathin hydrophilic shell. These ZES-SPIONs are free of Gd and show a high T1 contrast power. We demonstrate the potential of ZES-SPIONs in preclinical MRI and magnetic resonance angiography.

Thermogenic adipocytes promote HDL turnover and reverse cholesterol transport

Brown and beige adipocytes combust nutrients for thermogenesis and through their metabolic activity decrease pro-atherogenic remnant lipoproteins in hyperlipidemic mice. However, whether the activation of thermogenic adipocytes affects the metabolism and anti-atherogenic properties of high-density lipoproteins (HDL) is unknown. Here, we report a reduction in atherosclerosis in response to pharmacological stimulation of thermogenesis linked to increased HDL levels in APOE*3-Leiden.CETP mice. Both cold-induced and pharmacological thermogenic activation enhances HDL remodelling, which is associated with specific lipidomic changes in mouse and human HDL. Furthermore, thermogenic stimulation promotes HDL-cholesterol clearance and increases macrophage-to-faeces reverse cholesterol transport in mice. Mechanistically, we show that intravascular lipolysis by adipocyte lipoprotein lipase and hepatic uptake of HDL by scavenger receptor B-I are the driving forces of HDL-cholesterol disposal in liver. Our findings corroborate the notion that high metabolic activity of thermogenic adipocytes confers atheroprotective properties via increased systemic cholesterol flux through the HDL compartment.