|Substrate usage||13C NMR||Metabolism (I)|
|Body lipids||Substrat shift|
Analysis of substrate selection by 13C NMR spectroscopy
To ensure the continuous energy supply required for regular heart work, a permanent de novo synthesis of the spent ATP is absolutely essential. For this, primarily sugar and fatty acids are burned: a healthy heart normally fuels its energy production by one third from glucose and by two-thirds from palmitic acid oxidation. However, in several cardiac diseases in particular during development of cardiac hypertrophy, i.e. an overgrowth of the heart due to enhanced loading this normal ratio is shifted to an increased glucose utilization and a reduced oxidation of fatty acids.
Such substrate shifts can be smartly demonstrated by isotopomer analysis using 13C NMR spectroscopy, in which one makes use of the coupling patterns arising from neighbouring 13C nuclei. The principle of this method is schematically illustrated in the figure below and briefly described in the following.
The heart is supplied with substrates, at which the normal carbon isotope 12C is substituted by the naturally to less than 1% abundant isotope 13C (in contrast to 14C not radioactive) at suitable positions in the molecule. The exchanged atoms are marked in blue for glucose and in red for palmitate, respectively.
When these substrates are running through the normal metabolic pathways (first glycolysis, then the tricarboxylic acid (TCA) cycle also known as citric acid cycle), the 13C labels of glucose and palmitate, respectively, will end up at different positions of the glutamate carbon skeleton depending from the original substrate as well as the number of turns within the TCA cycle.
Who is interested in more facts about this, may trace by clicks on glucose and palmitate, respectively, in detail the fate of the molecule's label in metabolism and the formation of the individual glutamate isotopomers.
|The differing neighbourhood of 13C nuclei in the respective glutamate isotopomers is reflected in distinct coupling patterns within the 13C NMR spectra (see figure above): single labelling results in a singlet, double labelling in a doublet, and triple labelling in a doublet of doublet or rather a quartet. It is noteworthy, that on basis of the diverse coupling constants (abbreviated by J, see also theory) one can even distinguish, whether the double labelling is existent at C3+C4 (J34=34 Hz) or at C4+C5 (J45=51 Hz) of glutamate. Hence, it can be unequivocally determined from which carbon source the individual isotopomers originate, and thereby alterations in substrate metabolism can be detected (see below).|
Changes in substrate utilization
|The left figure shows the area of glutamate C4 from 13C NMR spectra of heart tissue taken from normal, healthy mice (bottom, WT=wildtype) and from mice, which are lacking the hemoprotein myoglobin (top, myo-/-). When comparing the signals from these spectra with the scheme of couplings shown above, one can retrieve in both cases the patterns for all of the four possible glutamate isotopomers. However, it is striking that the ratio of the signals, which originate from the metabolization of the two different substrates, differ clearly.|
While in the upper trace the patterns evoked by combustion of glucose are particularly eye-catching, in the lower spectrum the signals pointing to utilization of palmitate stick out. The quantitative analysis (displayed at the right) unambiguously shows that lack of myoglobin leads to a shift of the normal fuel utilization ratio of 1:3 (glucose:fatty acid) to an almost equal metabolization of both substrates a shift which is similarly observed during development of cardiac hypertrophy in man (see above).
Indeed myoglobin-deficient mice do surprisingly not exhibit any signs or cardiac disease despite the absence of the O2 carrier protein (O2=oxygen). This can be explained by the formation of a set of compensatory mechanisms, which are all aimed at buffering off the disturbance in O2 supply, such as increases in capillary density, coronary flow, coronary reserve, and hematocrit (cf. Gödecke et al, 1999). Thereby, the offer of O2 at the vascular site is enhanced, the O2 gradient from capillary to mitochondria increased, and consequently the diffusion of O2 from vessel to the place of consumption facilitated.
|In addition the substrate shift as detected by 13C NMR spectroscopy acts as a biochemical O2 saving mechanism at the consumer end: it is well known that for combustion of glucose noticeable less O2 is required as for combustion of fatty acids at generation of same amounts of ATP. Therefore, by increased utilization of the oxidative more favourable substrate, that is glucose, myoglobin-deficient hearts compensate the loss of the O2 carrier and storage protein myoglobin (for a detailed description and discussion of the respective experiments please have a look at Flögel et al, 2005).|
Own work about substrate metabolism
|A complete overview about our peer-reviewed publications of the last years can be found here. The references are linked with the PubMed abstracts of the National Library of Medicine. If your are interested in one of these papers, and you don't have online access to the respective journal, send us an email, so that we can provide you with the appropriate pdf-file.|
|Flögel U, Laussmann T, Gödecke A, Abanador N, Schäfers M, Fingas CD, Metzger S, Levkau B, Jacoby C, Schrader J.|
|Lack of myoglobin causes a switch in cardiac substrate selection.|
|Circ Res. 2005; 96: e68-75.|
|Flögel U, Willker W, Leibfritz D.|
|Determination of de novo synthesized amino acids in cellular proteins revisited by 13C NMR spectroscopy.|
|NMR Biomed. 1997; 10: 50-8.|
|Waclawczyk S, Buchheiser A, Flögel U, Radke TF, Kögler G.|
|In vitro differentiation of unrestricted somatic stem cells into functional hepatic-like cells displaying a hepatocyte-like glucose metabolism.|
|J Cell Physiol. 2010; 225: 545-54.|
|Flögel U, Leibfritz D.|
|Alterations in glial cell metabolism during recovery from chronic osmotic stress.|
|Neurochem Res. 1998; 23: 1553-61.|
|Willker W, Flögel U, Leibfritz D.|
|Ultra-high-resolved HSQC spectra of multiple 13C-labeled biofluids.|
|J Magn Reson. 1997; 125: 216-9.|
|Flögel U, Willker W, Engelmann J, Niendorf T, Leibfritz D.|
|Adaptation of cellular metabolism to anisosmotic conditions in a glial cell line, as assessed by 13C NMR spectroscopy.|
|Dev Neurosci. 1996; 18: 449-59.|