Mapping endocrine networks by stable isotope tracing

Abstract

Hormones regulate metabolic homeostasis through interlinked dynamic networks of proteins and small molecular weight metabolites, and state-of-the-art chemical technologies have been developed to decipher these complex pathways. Stable-isotope tracers have largely replaced radiotracers to measure flux in humans, building on advances in nuclear magnetic resonance spectroscopy and mass spectrometry. These technologies are now being applied to localise molecules within tissues. Radiotracers are still highly valuable both preclinically and in 3D imaging by positron emission tomography. The coming of age of vibrational spectroscopy in conjunction with stable-isotope tracing offers detailed cellular insights to map complex biological processes. Together with computational modelling, these approaches are poised to coalesce into multi-modal platforms to provide hitherto inaccessible dynamic and spatial insights into endocrine signalling.

Keywords: Flux; Magnetic resonance; Mass spectrometry; Positron emission tomography; Stable-isotope; Steroid; Vibrational spectroscopy.

Figure 1

Principles of isotope dilution at steady state. To quantify the rate of appearance of an endogenous molecule, known as the tracee, an isotopically labelled tracer is administered at a fixed rate. The tracer is a version of the tracee which is labelled with a radio or stable isotope. Samples of the biological pool are obtained, and the proportion of tracer to tracee is quantified. This value can be used to calculate the rate of appearance of the tracer; taking into account, their rates of clearance are equivalent. The Tracer:Tracee ratio is used to calculate the rate of appearance of the tracee based on a known infusion rate of tracer. The equation given requires the model to meet a number of assumptions and more complex models have been derived accounting for a series of variable parameters, for example numbers and sizes of pools, tracer recycling, measurement in steady state or non-steady state conditions, the degree of enrichment and priming, whole body or tissue specific sampling by arterio-venous sampling and the effect of the presence of an isotope on the fate of the labelled versus the unlabelled species — this list is not exhaustive and the reader is directed to [12] for further details. Tracer blue circles, Tracee open circles. AV= arterio-venous.

Figure 2

Imaging modalities allowing isotope tracing. (a) Fused ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PET/MR image demonstrating significant ¹⁸F-FDG uptake by brown adipose tissue bilaterally in the cervical and supraclavicular regions (arrows). ¹⁸F-FDG PET exploits the high rates of glucose uptake and glycolysis by human brown adipose tissue during cold activation. Following transport into tissues, ¹⁸F-FDG is converted to ¹⁸F-FDG-6-phosphate which cannot readily continue through the glycolytic pathway, thus is ‘trapped’ and accumulates in the cell, making this a very sensitive technique to quantify glucose uptake. The image was obtained from a subject housed at 16 °C for 3 h, following 2 h of cold exposure 75MBq of ¹⁸F-FDG was injected intravenously with this image taken an hour later. (b) Magnetic resonance image and ¹H-MR Spectroscopy spectra from a selected region of interest in the liver (square) obtained from a patient with type 2 diabetes mellitus, revealing high liver fat content. The water (∼4.7 ppm) and fat (∼2.3 ppm) peaks are shown on the spectra. T1 weighted images were obtained on a 1.5T GE MR scanner, spectra were obtained in a 30 × 30 × 30mm single voxel placed away from the major vessels using a PRESS sequence using a repetition time (TR) and echo time (TE) of 5000 and 40 ms, respectively. Thanks to Professor Mark Strachan and Dr Calum Gray, University of Edinburgh. (c) MS image collated using matrix-assisted laser desorption ionisation showing heatmaps representing [9,12,12-²H₃]-cortisol regenerated in regions of interest (ROI) of a mouse brain (male), including cortex, hippocampus and cerebellum (Cer), following steady state infusion of [9,11,12,12-²H₄]-cortisol and measured following derivatisation with Girard T. Full study reported in [59], but briefly, [9,11,12,12-²H₄]-cortisol is infused as a tracer to measure regeneration of cortisol by the enzyme 11β-hydroxysteroid dehydrogenase type 1. [9,11,12,12-²H₄]-cortisol is converted to [9,12,12-²H₃]-cortisone by the enzyme 11β-hydroxysteroid dehydrogenase type 2, and [9,12,12-²H₃]-cortisone is subsequently regenerated into active [9,12,12-²H₃]-cortisol by the type 1 isozyme. Thus cortisol generated de novo by the adrenal gland can be distinguished from that generated in other tissues (e.g. brain, liver, adipose) by 11β-hydroxysteroid dehydrogenase type 1. This tracer has been valuable in understanding the role of this enzyme in diseases of metabolic or cognitive dysfunction and in characterising it as a potential drug target. (d) Coherent anti-Stokes Raman spectroscopy showing the images of adipocytes treated with [2,2,4,6,6,7,21,21-²H]₈-(D₈)-corticosterone with or without MK571, an inhibitor of the steroid transporter ABCC1. Resonance of the carbon-hydrogen bonds abundant in lipid is represented in red; carbon deuterium resonance from D₈-corticosterone is represented in green and is more abundant with inhibition of export, notably in a distribution around the intracellular lipid droplets (scale bar, 20 μm). Reproduced under license number 5254281503812 [70]. Briefly, this approach has added value in understanding whether glucocorticoids pass into and out of cells by active and or passive transport. The historical view of the endocrine field has been that steroids passively traverse lipid membranes when travelling in and out of cells, but these recent studies suggest that ABCB1 and ABCC1 pumps have roles in actively exporting different glucocorticoids from cells where they are expressed.

Figure 3

The value of increasing the resolution of mass spectrometers on isotope distinction. The panels show the added value of increasing mass resolution on the ability to distinguish isotopologues of [9,12,12-²H₃] (D3F) and [9,11,12,12-²H₄]-cortisol (D4F) used in the studies of appearance of cortisol [11]. The simulation of mass resolution (Mr) settings of 1000 (typical of quadrupoles instruments), 80000 (typical of time of flight (ToF) instruments and 240000 and 480000 (achievable by Orbitrap® and Fourier transform ion cyclotron resonance instruments) were performed using https://www.protpi.ch/. Nominal masses of protonated ions of D3F and D4F are m/z 366 and m/z 367 and that of natural ¹³C-D3F, also 367. Using quadrupole instruments (Mr 1000), D3F containing one natural 13-carbon (m/z 367.234; green) cannot be resolved from D4F (m/z 367.242; blue). ToF instruments with Mr 80000 are also unable to resolve the two species. However, resolution can be achieved partially with Mr 240000 and fully baseline resolved at 480000, achievable using Orbitrap and Fourier transform ion cyclotron resonance instruments. Thus, the use of high resolution instruments can reduce background noise and remove the requirement for mathematical correction.