ex-vivo-hepatic-perfusion-through-the-portal-vein-in-mouse

<em>Ex Vivo</em> Hepatic Perfusion Through the Portal Vein in Mouse

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

University of Florida

Protein-ligand interaction is often characterized using polarization transfer by the intermolecular nuclear Overhauser effect (NOE). For such NOE experiments, hyperpolarization of nuclear spins presents the opportunity to increase the spin magnetization, which is transferred, by several orders of magnitude. Here, folic acid, a ligand of dihydrofolate reductase (DHFR), was hyperpolarized on (1)H spins using dissolution dynamic nuclear polarization (D-DNP). Mixing hyperpolarized ligand with protein resulted in observable increases in protein (1)H signal predominantly in the methyl group region of the spectra. Using (13)C single quantum selection in a series of one-dimensional spectra, the carbon chemical shift ranges of the corresponding methyl groups can be elucidated. Signals observed in these hyperpolarized spectra could be confirmed using 3D isotope filtered NOESY spectra, although the hyperpolarized spectra were obtained in single scans. By further correlating the signal intensities observed in the D-DNP experiments with the occurrence of short distances in the crystal structure of the protein-ligand complex, the observed methyl proton signals could be matched to the chemical shifts of six amino acids in the active site of DHFR-folic acid binary complex. These data demonstrate that (13)C chemical shift selection of protein resonances, combined with the intrinsic selectivity towards magnetization originating from the initially hyperpolarized spins, can be used for site specific characterization of protein-ligand interactions.

Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase.

The heart oxidizes fatty acids, carbohydrates, and ketone bodies inside the tricarboxylic acid (TCA) cycle to generate the reducing equivalents needed for ATP production. Competition between these substrates makes it difficult to estimate the extent of pyruvate oxidation. Previously, hyperpolarized pyruvate detected propionate-mediated activation of carbohydrate oxidation, even in the presence of acetate. In this report, the optimal concentration of propionate for the activation of glucose oxidation was measured in mouse hearts perfused in Langendorff mode. This study was performed with a more physiologically relevant perfusate than the previous work. Increasing concentrations of propionate did not cause adverse effects on myocardial metabolism, as evidenced by unchanged O consumption, TCA cycle flux, and developed pressures. Propionate at 1 mM was sufficient to achieve significant increases in pyruvate dehydrogenase flux (3×), and anaplerosis (6×), as measured by isotopomer analysis. These results further demonstrate the potential of propionate as an aid for the correct estimation of total carbohydrate oxidative capacity in the heart. However, liquid chromotography/mass spectroscopy-based metabolomics detected large changes (~30-fold) in malate and fumarate pool sizes. This observation leads to a key observation regarding mass balance in the TCA cycle; flux through a portion of the cycle can be drastically elevated without changing the O consumption.

A comprehensive analysis of myocardial substrate preference emphasizes the need for a synchronized fluxomic/metabolomic research design.

The kinase inhibitory domain of the cell cycle regulatory protein p27 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of  C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.

Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy.

Hyperpolarized carbon-13 magnetic resonance (HP-MR) is a new metabolic imaging method the does not use ionizing radiation. Due to the inherent chemical specificity of MR, not only tracer uptake but also downstream metabolism of the agent is detected in a straightforward manner. HP [2-C] dihydroxyacetone (DHA) is a promising new agent that directly interrogates hepatic glucose metabolism. DHA has three metabolic fates in the liver: glucose production, glycerol production and potential inclusion into triglycerides, and oxidation in the tricarboxylic acid cycle. Each pathway is regulated by flux through multiple enzymes. Using Duhamel's formula, the kinetics of DHA metabolism is modeled, resulting in estimates of specific reaction rate constants. The multiple enzymatic steps that control DHA metabolism make more simplified methods for extracting kinetic data less than satisfactory. The described modeling paradigm effectively identifies changes in metabolism between gluconeogenic and glycogenolytic models of hepatic function.

Kinetic Analysis of Hepatic Metabolism Using Hyperpolarized Dihydroxyacetone.

Metabolic flux modeling is a complementary technique to standard metabolomics/metabonomics as routinely practiced in biological systems. Here we outline carbon-13-based isotopomer analysis using nuclear magnetic resonance as the primary analytical technique. Isotopomer analysis produces relative rates of tricarboxylic acid cycle turnover. If these measures are normalized to O consumption, absolute rates can be inferred. The primary biological system targeted in this review is cell culture.

Nuclear Magnetic Resonance Measurement of Metabolic Flux Using C and H Signals.

Insulin resistance and altered hepatic mitochondrial function are central features of type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD), but the etiological role of these processes in disease progression remains unclear. Here we investigated the molecular links between insulin resistance, mitochondrial remodeling, and hepatic lipid accumulation.

Insulin resistance is mechanistically linked to hepatic mitochondrial remodeling in non-alcoholic fatty liver disease.

Ovine models of pregnancy have been used extensively to study maternal-fetal interactions and have provided considerable insight into nutrient transfer to the fetus. Ovine models have also been utilized to study congenital heart diseases. In this work, we demonstrate a comprehensive assessment of heart function and metabolism using a perinatal model of heart function with the addition of a [U-C]glucose as tracer to study central energy metabolism. Using nuclear magnetic resonance spectroscopy, and metabolic modelling, we estimate myocardial citric acid cycle turnover (normalized for oxygen consumption), substrate selection, and anaplerotic fluxes. This methodology can be applied to studying acute and chronic effects of hormonal signaling in future studies.

Application of Carbon-13 Isotopomer Analysis to Assess Perinatal Myocardial Glucose Metabolism in Sheep.

To determine whether deuterated water (HDO) generated from the metabolism of [ H ]glucose is a sensitive biomarker of cerebral glycolysis and oxidative flux.

Deuterated water imaging of the rat brain following metabolism of [ H ]glucose.

Imaging methods for hyperpolarized (HP) C agents must sample the evolution of signal from multiple agents with distinct chemical shifts within a very brief timeframe (typically < 1 min), which is challenging using conventional imaging methods. In this work, we compare two of the most commonly used HP spectroscopic imaging methods, spectral-spatial selective excitation and multi-echo chemical shift encoding (CSE, also referred to as IDEAL), for a typical preclinical HP [1-C]pyruvate imaging scan at 7 T. Both spectroscopic encoding techniques were implemented and validated in HP experiments imaging enzyme phantoms and the murine kidney. SNR performance of these two spectroscopic imaging approaches was compared in numerical simulations and phantom experiments using a single-shot flyback EPI readout for spatial encoding. With identical effective excitation angles, the SNR of images acquired with spectral-spatial excitations and CSE were found to be effectively equivalent.

Comparison of selective excitation and multi-echo chemical shift encoding for imaging of hyperpolarized [1-C]pyruvate.

Type II diabetes and pre-diabetes are widely prevalent among adults. Elevated serum glucose levels are commonly treated by targeting hepatic gluconeogenesis for downregulation. However, direct measurement of hepatic gluconeogenic capacity is accomplished only via tracer metabolism approaches that rely on multiple assumptions, and are clinically intractable due to expense and time needed for the studies. We previously introduced hyperpolarized (HP) [2-C]dihydroxyacetone (DHA) as a sensitive detector of gluconeogenic potential, and showed that feeding and fasting produced robust changes in the ratio of detected hexoses (6C) to trioses (3C) in the perfused liver. To confirm that this ratio is robust in the setting of treatment and hormonal control, we used ex vivo perfused mouse livers from BLKS mice (glucagon treated and metformin treated), and  mice. We confirm that the ratio of signal intensities of 6C to 3C in C nuclear magnetic resonance spectra post HP DHA administration is sensitive to hepatic gluconeogenic state. This method is directly applicable in vivo and can be implemented with existing technologies without the need for substantial modifications.

Hyperpolarized Dihydroxyacetone Is a Sensitive Probe of Hepatic Gluconeogenic State.

Metabolic flux analysis (MFA) combines experimental measurements and computational modeling to determine biochemical reaction rates in live biological systems. Advancements in analytical instrumentation, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), have facilitated chemical separation and quantification of isotopically enriched metabolites. However, no software packages have been previously described that can integrate isotopomer measurements from both MS and NMR analytical platforms and have the flexibility to estimate metabolic fluxes from either isotopic steady-state or dynamic labeling experiments. By applying physiologically relevant cardiac and hepatic metabolic models to assess NMR isotopomer measurements, we herein test and validate new modeling capabilities of our enhanced flux analysis software tool, INCA 2.0. We demonstrate that INCA 2.0 can simulate and regress steady-state C NMR datasets from perfused hearts with an accuracy comparable to other established flux assessment tools. Furthermore, by simulating the infusion of three different C acetate tracers, we show that MFA based on dynamic C NMR measurements can more precisely resolve cardiac fluxes compared to isotopically steady-state flux analysis. Finally, we show that estimation of hepatic fluxes using combined C NMR and MS datasets improves the precision of estimated fluxes by up to 50%. Overall, our results illustrate how the recently added NMR data modeling capabilities of INCA 2.0 can enable entirely new experimental designs that lead to improved flux resolution and can be applied to a wide range of biological systems and measurement time courses.

INCA 2.0: A tool for integrated, dynamic modeling of NMR- and MS-based isotopomer measurements and rigorous metabolic flux analysis.

The role of ketones in metabolic health has progressed over the past two decades, moving from what was perceived as a simple byproduct of fatty acid oxidation to a central player in a multiplicity of disease states. Previous work with hyperpolarized (HP) C has shown that ketone production can be detected when using precursors that labeled acetyl-CoA at the C1 position, often in tissues that are not normally recognized as ketogenic. Here, we assay metabolism of HP [2-C]pyruvate in the perfused mouse liver, a classic metabolic testbed where nutritional conditions can be precisely controlled. Livers perfused with long-chain fatty acids or the medium-chain fatty acid octanoate showed no evidence of ketogenesis in the C spectrum. In contrast, addition of dichloroacetate, a potent inhibitor of pyruvate dehydrogenase kinase, resulted in significant production of both acetoacetate and 3-hydroxybutyrate from the pyruvate precursor. This result indicates that ketones are readily produced from carbohydrates, but only in the case where pyruvate dehydrogenase activity is upregulated.

Article	Year
Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase. Journal of biomolecular NMR\| PubMed ID: 27189223	2016
A comprehensive analysis of myocardial substrate preference emphasizes the need for a synchronized fluxomic/metabolomic research design. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 28411229	2017
Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy. Angewandte Chemie (International ed. in English)\| PubMed ID: 28508552	2017
Kinetic Analysis of Hepatic Metabolism Using Hyperpolarized Dihydroxyacetone. Journal of chemical information and modeling\| PubMed ID: 30602117	2019
Nuclear Magnetic Resonance Measurement of Metabolic Flux Using C and H Signals. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 31127544	2019
Insulin resistance is mechanistically linked to hepatic mitochondrial remodeling in non-alcoholic fatty liver disease. Molecular metabolism\| PubMed ID: 33359401	2021
Application of Carbon-13 Isotopomer Analysis to Assess Perinatal Myocardial Glucose Metabolism in Sheep. Metabolites\| PubMed ID: 33466367	2021
Deuterated water imaging of the rat brain following metabolism of [ H ]glucose. Magnetic resonance in medicine\| PubMed ID: 33576535	2021
Comparison of selective excitation and multi-echo chemical shift encoding for imaging of hyperpolarized [1-C]pyruvate. Journal of magnetic resonance (San Diego, Calif. : 1997)\| PubMed ID: 33607386	2021
Hyperpolarized Dihydroxyacetone Is a Sensitive Probe of Hepatic Gluconeogenic State. Metabolites\| PubMed ID: 34357335	2021
INCA 2.0: A tool for integrated, dynamic modeling of NMR- and MS-based isotopomer measurements and rigorous metabolic flux analysis. Metabolic engineering\| PubMed ID: 34965470	2022
Detecting Hepatic Ketogenesis Using Hyperpolarized [2-C] Pyruvate. Frontiers in physiology\| PubMed ID: 35197867	2022

Mukundan Ragavan

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