Tissue-specific Short Chain Fatty Acid Metabolism and Slow Metabolic Recovery after Ischemia from Hyperpolarized NMR in Vivo

Abstract

Mechanistic details of mammalian metabolism in vivo and dynamic metabolic changes in intact organisms are difficult to monitor because of the lack of spatial, chemical, or temporal resolution when applying traditional analytical tools. These limitations can be addressed by sensitivity enhancement technology for fast in vivo NMR assays of enzymatic fluxes in tissues of interest. We apply this methodology to characterize organ-specific short chain fatty acid metabolism and the changes of carnitine and coenzyme A pools in ischemia reperfusion. This is achieved by assaying acetyl-CoA synthetase and acetyl-carnitine transferase catalyzed transformations in vivo. The fast and predominant flux of acetate and propionate signal into acyl-carnitine pools shows the efficient buffering of free CoA levels. Sizeable acetyl-carnitine formation from exogenous acetate is even found in liver, where acetyl-CoA synthetase and acetylcarnitine transferase activities have been assumed sequestered in different compartments. In vivo assays of altered acetate metabolism were applied to characterize pathological changes of acetate metabolism upon ischemia. Coenzyme pools in ischemic skeletal muscle are reduced in vivo even 1 h after disturbing muscle perfusion. Impaired mitochondrial metabolism and slow restoration of free CoA are corroborated by assays employing fumarate to show persistently reduced tricarboxylic acid (TCA) cycle activity upon ischemia. In the same animal model, anaerobic metabolism of pyruvate and tissue perfusion normalize faster than mitochondrial bioenergetics.Acetyl coenzyme A (acetyl-CoA) 2 is a central metabolite that connects metabolic paths such as fatty acid degradation and synthesis, cholesterol biosynthesis, glycolysis, and the TCA cycle (Fig. 1). Thus, acetyl-CoA is among the key molecules of energy and intermediary metabolism. Acetyl-CoA is generated by a number of enzymes in mammals including pyruvate dehydrogenase, ␤-ketothiolase, and ATP citrate-lyase (1). A less well-explored metabolic pathway forming acetyl-CoA in mammals is the acetyl-CoA synthetase (AceCS)-catalyzed catabolism of acetate (Fig. 1), which accounts for up to 10% of the energy expenditure in humans (2).Esterification of carboxylic acids is a common means of generating activated molecules for entrance into metabolic pathways and renders the CoA-activated metabolite largely membrane impermeable. Pools of acyl-CoA esters in different cellular compartments require tight regulation because of their metabolic activity and because of the signaling function of some CoA esters (3). Under normal conditions, free CoA is regenerated by the metabolism esters, whereas large amounts of CoA esters may accumulate under stress conditions (4). Sufficient pools of free CoA are ensured under these conditions by buffering acyl-CoA:CoA ratios through transesterifications of acylCoA with carnitine. Carnitine also provides a shuttle for the flux of CoA-activated metabolites between intracellular compartments (5). Transesterification between CoA and carnitine est...

Direct noninvasive estimation of myocardial tricarboxylic acid cycle flux in vivo using hyperpolarized 13C magnetic resonance

“…The acetylCoA resonance previously reported at 202 ppm after a 90°e xcitation in the mouse heart following the injection of hyperpolarized [1-13 C]acetate could not be detected in the present study [36]. Detectable quantities of 13 C-labeled glutamate could be expected since 60% of the 14 C activity measured 2 min after the injection of 14 C-labeled acetate was associated with glutamate while a mere 8% was found to come from TCA cycle metabolites [57].…”

“…In this study, the estimated maximum plasma concentration at the end of the bolus injection was between 3 and 15 mM, which immediately decayed after the bolus injection. In previous metabolic studies, 45 min-long acetate infusions have been used, leading to plasma concentrations up to 10 mM [16,51], and no adverse effects were reported in hyperpolarized MR studies with comparable doses of 13 C-acetate [35,36,[52][53][54]. In the present study, all monitored physiological parameters remained stable before and after injection (Table 1), and no adverse effects were observed following acetate administration.…”

“…In contrast to earlier investigations in cardiac muscle performed at 2.35 T or 3 T [36,52,54], the increased spectral resolution obtained at 9.4 T allowed for the direct observation of the [5-13 C]citrate resonance, and illustrates the benefit and need for high field. The delayed observation of citrate with respect to acetylcarnitine is consistent with the role of carnitine as a shuttle for acetyl groups across the mitochondrial membrane, and as a mitochondrial buffer for excess acetylCoA.…”

“…A study of hyperpolarized [1-13 C]acetate metabolism in resting rat skeletal muscle showed that the rate of acetylcarnitine 13 C labeling can be directly translated to the metabolic flux through ACS [35]. However, despite the fact that [1-13 C]acetylCoA was detected in the mouse heart and liver following the injection of hyperpolarized [1-13 C]acetate [36], 13 C label incorporation from acetate into myocardial TCA cycle intermediates has to date not been reported in vivo. This is most likely due to their low concentration combined with their spectral proximity to the large 13 C resonance of the injected precursor.…”

Current state-of-the-art hyperpolarized 13 C-acetate-to-acetylcarnitine imaging is not indicative of the altered balance between glucose and fatty acid utilization associated with diabetes

“…This difference in short‐chain fatty acid metabolism is likely originating from the intraorgan difference in acetyl‐CoA synthetase isoform distribution in tissue (Jensen et al. 2009). …”