Mahon L. Maguire

Environmental pollution and diabetes: a neglected association

Using cross-sectional data from the 1999–2002 US National Health and Examination Survey, Duk-Hee Lee and colleagues reported a strong correlation between insulin resistance and serum concen trations of persistent organic pollutants, especially for organochlorine com pounds. This result was a surprise for many people working in diabetes research, because most studies to date have focused on the eff ects of genetics and the westernisation of dietary habits and lifestyle, while ignoring the potential eff ect of xenobiotics. Nevertheless, as discussed by Porta, an increasing number of reports suggest that chronic dietary exposure to, and accumulation of, low concentrations of environmental pollutants within the body might also be associated with diabetogenesis. Ignoring the potential eff ects of xenobiotics therefore risks ignoring a potentially crucial component in the study of type 2 diabetes, obesity, and the metabolic syndrome. Lee and co-workers note two important fi ndings related to obesity. The fi rst was that the expected association between obesity and diabetes was absent in people with low concentrations of persistent organic pollutants in their blood. The second was that the association between obesity and diabetes became stronger as the concentrations of such pollutants in the blood increased. Interestingly, this report is not the fi rst time that the presence of persistent organic pollutants and other organochlorine compounds in blood have been associated with diabetes. Rylander and colleagues analysed the concentrations of 2,2’,4,4’,5,5’-hexachlorobiphenyl (CB-153) and 1,1dichloro-2,2-bis (p-chlorophenyl) -ethylene (p,p’-DDE, a breakdown product of DDT) in the serum of 196 men and 184 women in a fi shing community in Sweden. Both compounds showed a strong association with the occurrence of diabetes. Much the same results were seen in a population-based study in Belgium. Studies of US Air Force veterans of the Vietnam War exposed to Agent Orange also suggest an adverse relation between dioxin exposure and symptoms of diabetes. The potential of xenobiotics to disrupt glucose and lipid metabolism in mammals is a well-developed theory in toxicology. Indeed, many of the early toxicity responses in animal studies with a range of pollutants note glucosuria, dyslipidaemia, increased gluconeogenesis, and fatty liver. The versatility of high-throughput screening in metabolomics and metabonomics is an especially useful way of monitoring metabolic changes caused by disease or exposure to toxicants (eg, heavy metals) in animal models. Although most studies have tested acute exposure (ie, less than 2 weeks), in the few chronic exposure studies (ie, more than 3 months), glucosuria was recorded as long-term eff ect. This result suggests that diabetes could be exacerbated by chronic exposure to xenobiotics that disrupt normal metabolism of glucose and lipids. Although correlation does not necessarily prove the existence of a causal relation, biologically plausible ex plan ations exist for this association. For instance, dioxin-like compounds exert their eff ects through binding to the aryl-hydrocarbon receptor. This receptor is thought to antagonise peroxisome proliferator-activated receptors. One plausible hypothesis, there fore, is that the aryl-hydrocarbon receptor promotes dia beto genesis by antagonism of peroxisome proliferator-activated receptors. However, no data are avail able to support this See Correspondence page 302

Omic analyses unravels global molecular changes in the brain and liver of a rat model for chronic sake (Japanese alcoholic beverage) intake

The effects of chronic administration of Sake (Japanese alcoholic beverage, Nihonshu) on brain and liver of female F334 (Fisher) rats were surveyed via global omic analyses using DNA microarray, 2‐DE, and proton nuclear magnetic resonance. Rats weaned at 4 wk of age were given free access to Sake (15% alcohol), instead of water. At 13 months of age, and 24 h after withdrawal of Sake supply, rats were sacrificed, and the whole brain and liver tissues dissected for analyses. In general, molecular changes in brain were found to be less than those in liver. Transcriptomics data revealed 36 and 9, and 80 and 62 up‐ and down‐regulated genes, in the brain and liver, respectively, with binding and catalytic activity gene categories the most prominently changed. Results suggested Sake‐induced fragility of brain and liver toxicity/damage, though no significant abnormalities in growth were seen. At protein level, a striking decrease was found in the expression of NADH dehydrogenase (ubiquinone) Fe‐S protein 1 in brain, suggesting attenuation of mitochondrial metabolism. In liver, results again suggested an attenuation of mitochondrial function and, in addition, glycoproteins with unknown function were induced at protein and gene levels, suggesting possible changes in glycoprotein binding in that organ. Metabolomic analysis of brain revealed significant increases in valine, arginine/ornithine, alanine, glutamine, and choline with decreases in isoleucine, N‐acetyl aspartate, taurine, glutamate, and gamma aminobutyric acid. Our results provide a detailed inventory of molecular components of both brain and liver after Sake intake, and may help to better understand effects of chronic Sake drinking.

Resolving Fine Cardiac Structures in Rats with High-Resolution Diffusion Tensor Imaging.

Cardiac architecture is fundamental to cardiac function and can be assessed non-invasively with diffusion tensor imaging (DTI). Here, we aimed to overcome technical challenges in ex vivo DTI in order to extract fine anatomical details and to provide novel insights in the 3D structure of the heart. An integrated set of methods was implemented in ex vivo rat hearts, including dynamic receiver gain adjustment, gradient system scaling calibration, prospective adjustment of diffusion gradients, and interleaving of diffusion-weighted and non-diffusion-weighted scans. Together, these methods enhanced SNR and spatial resolution, minimised orientation bias in diffusion-weighting, and reduced temperature variation, enabling detection of tissue structures such as cell alignment in atria, valves and vessels at an unprecedented level of detail. Improved confidence in eigenvector reproducibility enabled tracking of myolaminar structures as a basis for segmentation of functional groups of cardiomyocytes. Ex vivo DTI facilitates acquisition of high quality structural data that complements readily available in vivo cardiac functional and anatomical MRI. The improvements presented here will facilitate next generation virtual models integrating micro-structural and electro-mechanical properties of the heart.

Efficient gradient calibration based on diffusion MRI.

To propose a method for calibrating gradient systems and correcting gradient nonlinearities based on diffusion MRI measurements.

Temporal accumulation and localization of isoflurane in the C57BL/6 mouse and assessment of its potential contamination in 19 F MRI with perfluoro-crown-ether-labeled cardiac progenitor cells at 9.4 Tesla.

To assess the uptake, accumulation, temporal stability, and spatial localization of isoflurane (ISO) in the C57BL/6 mouse, and to identify its potential interference with the detection of labeled cardiac progenitor cells using 19F MRI/MR spectroscopy (MRS).

High-Resolution Magnetic Resonance Imaging of the Regenerating Adult Zebrafish Heart.

The adult zebrafish is a well-established model for studying heart regeneration, but due to its tissue opaqueness, repair has been primarily assessed using destructive histology, precluding repeated investigations of the same animal. We present a high-resolution, non-invasive in vivo magnetic resonance imaging (MRI) method incorporating a miniature respiratory and anaesthetic perfusion set-up for live adult zebrafish, allowing for visualization of scar formation and heart regeneration in the same animal over time at an isotropic 31 µm voxel resolution. To test the method, we compared well and poorly healing cardiac ventricles using a transgenic fish model that exhibits heat-shock (HS) inducible impaired heart regeneration. HS-treated groups revealed persistent scar tissue for 10 weeks, while control groups were healed after 4 weeks. Application of the advanced MRI technique allowed clear discrimination of levels of repair following cryo- and resection injury for several months. It further provides a novel tool for in vivo time-lapse imaging of adult fish for non-cardiac studies, as the method can be readily applied to image wound healing in other injured or diseased tissues, or to monitor tissue changes over time, thus expanding the range of questions that can be addressed in adult zebrafish and other small aquatic species.

Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.

Purpose To a) achieve cardiac 19F-Magnetic Resonance Imaging (MRI) of perfluoro-crown-ether (PFCE) labeled cardiac progenitor stem cells (CPCs) and bone-derived bone marrow macrophages, b) determine label concentration and cellular load limits, and c) achieve spectroscopic and image-based quantification. Methods Theoretical simulations and experimental comparisons of spoiled-gradient echo (SPGR), rapid acquisition with relaxation enhancement (RARE), and steady state at free precession (SSFP) pulse sequences, and phantom validations, were conducted using 19F MRI/Magnetic Resonance Spectroscopy (MRS) at 9.4 T. Successful cell labeling was confirmed using flow cytometry and confocal microscopy. For CPC and macrophage concentration quantification, in vitro and post-mortem cardiac validations were pursued with the use of the transfection agent FuGENE. Feasibility of fast imaging is demonstrated in murine cardiac acquisitions in vivo, and in post-mortem murine skeletal and cardiac applications. Results SPGR/SSFP proved favorable imaging sequences yielding good signal-to-noise ratio values. Confocal microscopy confirmed heterogeneity of cellular label uptake in CPCs. 19F MRI indicated lack of additional benefits upon label concentrations above 7.5–10 mg/ml/million cells. The minimum detectable CPC load was ~500k (~10k/voxel) in two-dimensional (2D) acquisitions (3–5 min) using the butterfly coil. Additionally, absolute 19F based concentration and intensity estimates (trifluoroacetic-acid solutions, macrophages, and labeled CPCs in vitro and post-CPC injections in the post-mortem state) scaled linearly with fluorine concentrations. Fast, quantitative cardiac 19F-MRI was demonstrated with SPGR/SSFP and MRS acquisitions spanning 3–5 min, using a butterfly coil. Conclusion The developed methodologies achieved in vivo cardiac 19F of exogenously injected labeled CPCs for the first time, accelerating imaging to a total acquisition of a few minutes, providing evidence for their potential for possible translational work.

Magnetic resonance imaging of the regenerating neonatal mouse heart

We present longitudinal magnetic resonance imaging (MRI) of neonatal mouse hearts during the first three weeks following coronary artery ligation to mimic heart attack. We confirm heart regeneration in individual animals injured on post-natal day 1 (P1) while those injured on P7 show the adult response of fibrosis, scarring and impaired heart performance. We document heart growth and development of the principal functional cardiac parameters, and also remodeling during tissue regeneration as compared to fibrosis when imaging repeatedly up to 21 days after myocardial infarction (MI). We reveal compensatory changes in cardiac function with the restoration of tissue and resolution of injury for the P1 cohort and sustained injury responses for the P7 cohort. This study resolves the controversy surrounding neonatal mouse heart regeneration and establishes a functional platform for live capture of the regenerative process and for the future testing of genetic or therapeutic interventions.

Preclinical Cardiac In Vivo Spectroscopy

Heart disease, and specifically heart failure, result from a complex interaction of genetics, metabolism, and physical insult/ischaemia that lead to changes in myocardial metabolism, gene expression, hypertrophy, cell death, or scarring. Magnetic resonance spectroscopy has been employed as a key tool for probing myocardial metabolism as it uniquely provides a nondestructive, noninvasive, method to probe metabolism without the need for additional radioactive probes. Many surgical and genetic animal models, which mimic the characteristics of human cardiac disease, have become established enabling researchers to study the physical and metabolic changes involved at a level of detail that is typically not possible in the clinic. In the preclinical context, a variety of pulse sequences, radio-frequency coils, and physical set-ups have been used to measure magnetic resonance spectra in order to study myocardial metabolism both ex vivo and in vivo. This chapter provides an overview of the current state of the art, and the animal models and biological systems to which they have been applied.

Mass Spectrometry-Based Metabolomics. Sample Preparation, Data Analysis, and Related Analytical Approaches

Metabolomics refers to the study of small-molecule metabolites (e.g. fats, sugars, nuclear acids) within biological samples such as cells, tissues, biofluids or even whole organisms. It has been found to be applicable to a wide range of fields, including the study of gene function, toxicology, plant sciences, environmental analysis, cancer, clinical diagnostics, nutrition and the discrimination of organism genotypes to name but a few. The approach combines high-throughput sample analysis with computer-assisted multivariate pattern-recognition techniques. A major challenge in metabolomics is to address the extremely diverse and complex nature of the subject matter and both past and future progress in the field depends in large part on the use and evolution of analytical techniques and instrumentation, especially mass spectrometry. This chapter therefore focuses on outlining and discussing current thinking behind the most commonly used analytical methodologies as well as associated multivariate data processing techniques.