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Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.
Carotid body glomus cells mediate essential reflex responses to arterial blood hypoxia. They are dopaminergic and secrete growth factors that support dopaminergic neurons, making the carotid body a potential source of patient-specific cells for Parkinson's disease therapy. Like adrenal chromaffin cells, which are also hypoxia-sensitive, glomus cells are neural crest-derived and require the transcription factors Ascl1 and Phox2b; otherwise, their development is little understood at the molecular level. Here, analysis in chicken and mouse reveals further striking molecular parallels, though also some differences, between glomus and adrenal chromaffin cell development. Moreover, histology has long suggested that glomus cell precursors are 'émigrés' from neighbouring ganglia/nerves, while multipotent nerve-associated glial cells are now known to make a significant contribution to the adrenal chromaffin cell population in the mouse. We present conditional genetic lineage-tracing data from mice supporting the hypothesis that progenitors expressing the glial marker proteolipid protein 1, presumably located in adjacent ganglia/nerves, also contribute to glomus cells. Finally, we resolve a paradox for the 'émigré' hypothesis in the chicken - where the nearest ganglion to the carotid body is the nodose, in which the satellite glia are neural crest-derived, but the neurons are almost entirely placode-derived - by fate-mapping putative nodose neuronal 'émigrés' to the neural crest.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20-95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species.
DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.
Campylobacter jejuni is a leading cause of bacterially derived gastroenteritis. A previous mutant screen demonstrated that the heme uptake system (Chu) is required for full colonization of the chicken gastrointestinal tract. Subsequent work identified a PAS domain-containing regulator, termed HeuR, as being required for chicken colonization. Here we confirm that both the heme uptake system and HeuR are required for full chicken gastrointestinal tract colonization, with the heuR mutant being particularly affected during competition with wild-type C. jejuni Transcriptomic analysis identified the chu genes-and those encoding other iron uptake systems-as regulatory targets of HeuR. Purified HeuR bound the chuZA promoter region in electrophoretic mobility shift assays. Consistent with a role for HeuR in chu expression, heuR mutants were unable to efficiently use heme as a source of iron under iron-limiting conditions, and mutants exhibited decreased levels of cell-associated iron by mass spectrometry. Finally, we demonstrate that an heuR mutant of C. jejuni is resistant to hydrogen peroxide and that this resistance correlates to elevated levels of catalase activity. These results indicate that HeuR directly and positively regulates iron acquisition from heme and negatively impacts catalase activity by an as yet unidentified mechanism in C. jejuni IMPORTANCE: Annually, Campylobacter jejuni causes millions of gastrointestinal infections in the United States, due primarily to its ability to reside within the gastrointestinal tracts of poultry, where it can be released during processing and contaminate meat. In the developing world, humans are often infected by consuming contaminated water or by direct contact with livestock. Following consumption of contaminated food or water, humans develop disease that is characterized by mild to severe diarrhea. There is a need to understand both colonization of chickens, to make food safer, and colonization of humans, to better understand disease. Here we demonstrate that to efficiently colonize a host, C. jejuni requires iron from heme, which is regulated by the protein HeuR. Understanding how HeuR functions, we can develop ways to inhibit its function and reduce iron acquisition during colonization, potentially reducing C. jejuni in the avian host, which would make food safer, or limiting human colonization.
Copyright © 2016 Johnson et al.
Radiation force-based elasticity imaging is currently being investigated as a possible diagnostic modality for a number of clinical tasks, including liver fibrosis staging and the characterization of cardiovascular tissue. In this study, we evaluate the relationship between peak displacement magnitude and image quality and propose using a Bayesian estimator to overcome the challenge of obtaining viable data in low displacement signal environments. Displacement data quality were quantified for two common radiation force-based applications, acoustic radiation force impulse imaging, which measures the displacement within the region of excitation, and shear wave elasticity imaging, which measures displacements outside the region of excitation. Performance as a function of peak displacement magnitude for acoustic radiation force impulse imaging was assessed in simulations and lesion phantoms by quantifying signal-to-noise ratio (SNR) and contrast-to-noise ratio for varying peak displacement magnitudes. Overall performance for shear wave elasticity imaging was assessed in ex vivo chicken breast samples by measuring the displacement SNR as a function of distance from the excitation source. The results show that for any given displacement magnitude level, the Bayesian estimator can increase the SNR by approximately 9 dB over normalized cross-correlation and the contrast-to-noise ratio by a factor of two. We conclude from the results that a Bayesian estimator may be useful for increasing data quality in SNR-limited imaging environments.
Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.
Epithelial-to-mesenchymal transition (EMT) of endocardial cells is a critical initial step in the formation of heart valves. The collagen gel in vitro model has provided significant information on the role of growth factors regulating EMT but has not permitted investigation of mechanical factors. Therefore we sought to develop a system to probe the effects of mechanical inputs on endocardial EMT by incorporating hyaluronic acid (HA), the primary component of endocardial cushions in developing heart valves, into the gel assay. This was achieved using a combination collagen and crosslinkable methacrylated HA hydrogel (Coll-MeHA). Avian atrioventricular canal explants on Coll-MeHA gels showed increased numbers of transformed cells. Analysis of the mechanical properties of Coll-MeHA gels shows that stiffness does not directly affect EMT. Hydrogel deformation from the beating myocardium of explants directly led to higher levels of regional gel deformation and larger average strain magnitudes associated with invaded cells on Coll-MeHA gels. Inhibition of this contraction reduced EMT on all gel types, although to a lesser extent on Coll-MeHA gels. Using the system we have developed, which permits the manipulation of mechanical factors, we have demonstrated that active mechanical forces play a role in the regulation of endocardial EMT.
Copyright © 2013 Elsevier Ltd. All rights reserved.
Valvular Interstitial Cells (VICs) are a common substrate for congenital and adult heart disease yet the signaling mechanisms governing their formation during early valvulogenesis are incompletely understood. We developed an unbiased strategy to identify genes important in endocardial epithelial-to-mesenchymal transformation (EMT) using a spatial transcriptional profile. Endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo an EMT to yield VICs. RNA sequencing (RNA-seq) analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick and mouse embryos at comparable stages of development (chick HH18; mouse E11.0) was performed. EMT occurs in the AVC and OFT cushions, but not VEN at this time. 198 genes in the chick (n=1) and 105 genes in the mouse (n=2) were enriched 2-fold in the cushions. Gene regulatory networks (GRN) generated from cushion-enriched gene lists confirmed TGFβ as a nodal point and identified NF-κB as a potential node. To reveal previously unrecognized regulators of EMT four candidate genes, Hapln1, Id1, Foxp2, and Meis2, and a candidate pathway, NF-κB, were selected. In vivo spatial expression of each gene was confirmed by in situ hybridization and a functional role for each in endocardial EMT was determined by siRNA knockdown in a collagen gel assay. Our spatial-transcriptional profiling strategy yielded gene lists which reflected the known biology of the system. Further analysis accurately identified and validated previously unrecognized novel candidate genes and the NF-κB pathway as regulators of endocardial cell EMT in vitro.
Copyright © 2013 Elsevier Ltd. All rights reserved.
INTRODUCTION - Muscle growth and regeneration are processes closely associated with proliferation, differentiation, and apoptosis of muscle cells. Death-associated protein 1 (DAP1) has been identified as a negative regulator of autophagy. Little is known about the function of DAP1 in the regulation of myogenesis and satellite cells.
METHODS - Chicken satellite cells were transfected with DAP1 cloned into the pCMS-enhanced green fluorescent protein vector or pcDNA3.1 vector, or a small interference RNA against the endogenous DAP1 gene. The cells were assayed for proliferation, differentiation, and apoptosis.
RESULTS - The overexpression of DAP1 increased proliferation, differentiation, and myotube diameter, but it had no effect on satellite cell apoptosis. In contrast, knockdown of DAP1 significantly decreased proliferation, differentiation, and number of nuclei per myotube, and it increased apoptosis of the cells.
CONCLUSION - DAP1 is required for regulating myogenesis and apoptosis of satellite cells, which may affect muscle mass accretion and regeneration, and ameliorate muscle sarcopenia.
Copyright © 2013 Wiley Periodicals, Inc.
The spatial distribution of molecular signals within cells is crucial for cellular functions. Here, as a model to study the polarized spatial distribution of molecular activities, we used cells on micropatterned strips of fibronectin with one end free and the other end contacting a neighbouring cell. Phosphoinositide 3-kinase and the small GTPase Rac display greater activity at the free end, whereas myosin II light chain and actin filaments are enriched near the intercellular junction. Phosphoinositide 3-kinase and Rac polarization depend specifically on the N-cadherin-p120 catenin complex, whereas myosin II light chain and actin filament polarization depend on the N-cadherin-β-catenin complex. Integrins promote high phosphoinositide 3-kinase/Rac activities at the free end, and the N-cadherin-p120 catenin complex excludes integrin α5 at the junctions to suppress local phosphoinositide 3-kinase and Rac activity. We hence conclude that N-cadherin couples with distinct effectors to polarize phosphoinositide 3-kinase/Rac and myosin II light chain/actin filaments in migrating cells.