Transfer of Glucose Hydrogens via Acetyl-CoA, Malonyl-CoA and NADPH to Fatty Acids during de novo Lipogenesis

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Introduction:
There is currently high interest in the measurement of de novo lipogenesis (DNL) to better understand its role in the dyslipidemia of diseases such as Type 2 diabetes and fatty liver disease (1-3). Fractional DNL rates can be measured from incorporation of deuterated water ( 2 H 2 O) into fatty acids (4, 5)an inexpensive and simple method that can be applied to humans, animal models and cell cultures. 2 H enrichment of fatty acids from 2 H 2 O is conventionally measured by massspectrometry (MS), where all the fatty acid chain hydrogens are considered as a single traceable entity (4). While this provides amplification of the m+1 signal arising from 2 H incorporation, it does not resolve the carbon-bound fatty acid hydrogens according to their metabolic sources (see Figure 1). It was reported that in rats, about 30% of plasma triglyceride palmitate hydrogens had not exchanged with 2 H-body water (6, 7) while for palmitate derived from cultured cells, this nonexchanged fraction was even higher (8). Therefore for DNL measurement, the 2 H-enrichment of fatty acids measured by MS needs to be corrected by a pre-determined factor related to the number of deuterium atoms that were incorporated per molecule of fatty acid, referred to as N (6).
Stoichiometric 2 H-enrichment of fatty acids from 2 H 2 O is conditional on full exchange between the hydrogens of water and those of the acetyl-CoA methyls, the malonyl-CoA methylenes, and the reducing hydrogen of NADPH. A less than theoretical 2 H-enrichment of the fatty acids implies that hydrogen exchange is incomplete, but to what degree this occurs for each of the metabolic precursors is not known. To address this, we provided mice with [U-2 H 7 ]glucose and performed 2 H NMR analysis of liver triglyceride (5, 9,10) to determine the extent to which the 2 H were transferred into positions 2, 3 and the terminal methyl hydrogens of hepatic fatty acids: each position reflecting 2 H transfer from glucose via malonyl-CoA, NADPH and acetyl-CoA, respectively. Our data indicate that there was a substantial transfer of glucose hydrogen to newlysynthesized fatty acids via NADPH, corresponding to a limited exchange with water hydrogen, but relatively low transfer via malonyl-CoA and acetyl-CoA intermediates indicating extensive hydrogen exchange at these loci of the DNL pathway. by guest, on March 6, 2020 www.jlr.org Downloaded from pyrazine standard signal. These were obtained as mean values from a set of seven liver triglyceride samples obtained from mice administered with 2 H 2 O. For each sample, a spectrum was acquired with the described parameters and immediately followed by a spectrum acquired under the same parameters with the exception of the acquisition time and pulse delay, which were set to 1 second and 8 seconds, respectively. The correction factors for the 2 H signals in fatty acid position 2, position 3 and methyl hydrogens were 0.51, 0.52 and 0.88, respectively. For 13 C isotopomer analysis by 13 C NMR, dried triglyceride samples were dissolved in 0.2 ml 99.96% enriched CDCl 3 (Sigma-Aldrich) and placed in 3 mm NMR tubes. 13 C NMR spectra were acquired at 150.8 MHz with an Agilent V600 spectrometer equipped with a 3 mm broadband probe. Spectra were acquired with a 70° pulse, an acquisition time of 2.5 seconds, and a 0.5 second pulse delay. For each spectrum, 2,000-4,000 fid were collected.

Quantification of triglyceride positional 2 H-and 13 C-enrichments:
Positional 2 H-enrichments of triglyceride fatty acids were quantified by analysis of 1 H and 2 H NMR triglyceride spectra as previously described (5, 10). From the methyl and carboxyl 13 C NMR resonances, positional 13 Cenrichments of fatty acids were estimated from the ratio of 13 C-13 C-spin-coupled doublet signals (representing positional isotopomers derived from [1,2-13 C 2 ]acetyl-CoA) to the singlet signal, representing the 1.11% natural abundance 13 C (see Supplementary Figure 1). From the methyl singlet and doublet 13 C NMR signals, the 13 C-enrichment of fatty acids from [1,2-13 C 2 ]acetyl-CoA in the methyl position was calculated as follows: Excess 13 C-enrichment of fatty acid methyls (%) = Methyl D/Methyl S × 1.11 Where Methyl D and Methyl S are the doublet and singlet components, respectively, of the 13 Csignal of the fatty acid terminal methyls and 1.11 represents the background 13 C-enrichment (%).
Assuming that fatty acid enrichment from [1,2-13 C 2 ]acetyl-CoA via elongation was limited to position 1 and 2 carbons, enrichment of carbon 3 were assumed to be equivalent to those of the terminal methyl carbon.
From analysis of the fatty acid singlet and doublet carboxyl 13 C-signals, excess enrichment of the position 1 of fatty acids from [1,2-13 C 2 ]acetyl-CoA was estimated as follows: Position 1 Excess 13 C-enrichment (%) = D/ S × 1.11 Where D and S are the summed doublet and singlet components, respectively, of the 13 Ccarboxyl resonances and 1.11 represents the background 13 C-enrichment (%). Excess enrichment of the fatty acid position 2 carbon was assumed to be equal to that of position 1.   (Table 1).
Normalizing the fatty acid enrichment from [U-2 H 7 ]glucose to that of [U-13 C 6 ]glucose provides a measure of the fractional retention of the [U-2 H 7 ]glucose 2 H atoms in a given position (Table 2).
These data reveal that for those fatty acids that were synthesized from exogenous glucose, far more 2 H was transferred into the position 3 hydrogens compared to either position 2 or the terminal methyl hydrogens. This indicates a greater exchange of 2 H and water hydrogens during the conversion of [U-2 H 7 ]glucose to acetyl-CoA and malonyl-CoA compared to hydrogen transfer via PPP oxidation and NADPH. Only one of the fatty acid position 2 hydrogens is derived from malonyl-CoA, the other originates from body water. Therefore, based on the observed fatty acid position 2 2 H/ 13 C enrichment ratio of 6%, we can infer that the 2 H/ 13 C enrichment ratio of the malonyl-CoA precursor was 12%. This is similar to the 14% estimated for the initial acetyl-CoA pool recruited by fatty acid synthase (FAS).
We performed an additional a set of studies where [U- 13  label, fatty acids were enriched in position 3 while enrichment of position 2 was not detectable and the terminal methyl had a vestigial 2 H signal. Given the substantial transfer of glucose hydrogens into the position 3 of fatty acid relative to position 2, it might be expected that for mice administered with 2 H 2 O, enrichment of fatty acid position 3 would also be less than that of position 2. However, as shown by Figure 2c, the intensities of position 2 and 3 signals were similar, and there was no significant difference between the 2 H-enrichments quantified for each site (1.79 ± 0.19% and 1.81± 0.19% for positions 2 and 3, respectively).

Discussion
Since the pioneering studies of Beylot and co-workers (4, 6), 2 H 2 O has been extensively used as a  Table 2). This indicates that under our study conditions, there was no additional loss of 2 H from glucose position 1 compared to position 3, for example by exchange of glucose-6-phosphate with fructose-6-phosphate and mannose-6-phosphate (15). Direct studies of intracellular NADPH enrichment from 2 H 2 O report a greater degree of exchange between the reducing hydrogen and body water than might be expected based on our observations (16). One explanation for this is that the PPP is not the sole source of intracellular NADPH, as illustrated by Figure 3. In mitochondria, NADPH can be generated from NADH by nicotinamide nucleotide transhydrogenase. In mitochondria as well as cytosol, NADPH can also be generated via NADP + -malic enzyme and NADP + -isocitrate dehydrogenase. In all of these cases, the likelihood that the hydrogen that was transferred to NADP + had previously exchanged with body water is high. For NADPH formed via the NADP +malic enzyme, the reducing hydrogen originates from hydrogen 2 of malate, which in turn originated from water during the hydration of fumarate. If the malate is metabolized via the Krebs cycle to citrate and isocitrate, this hydrogen is transferred to NADPH via NADP + -isocitrate dehydrogenase. The bulk of mitochondrial NADH hydrogens are also derived from Krebs cycle intermediates such as malate, whose hydrogens are highly exchanged with those of water. In addition, NADPH and water hydrogens may also be exchanged via NADP + -linked redox enzymes (16,17 conditions, hepatic glucose-6-phosphate becomes highly enriched with 2 H in all positions due to extensive cycling between glucose-6-phosphate and gluconeogenic precursors (20, 21). Under these conditions, NADPH derived from PPP oxidation of glucose-6-phosphate will also become highly enriched with 2 H and this will be transferred to the respective fatty acid positions.

Implications for estimating the number of deuterium atoms incorporated per fatty acid (N): Under
our study conditions, the number of deuterium atoms incorporated per fatty acid molecule (N) appears to approach the theoretical value (i.e. 31 for palmitate). Therefore in this instance, no correction needs to be applied to the observed fatty acid 2 H enrichment from 2 H 2 O. This differs from previous studies in rats, where N was estimated to be 21 and 22 out of 31 (68% and 71% of the theoretical value) by mass isotopomer distribution analysis (MIDA) for plasma and liver triglyceride palmitate, respectively (6, 7). To our knowledge, N has not been determined in the mouse by MIDA, but given its smaller size and higher basal metabolic rate, it might be expected to be higher compared to the rat. The mice in our study also ingested significant amounts of fructose, a sugar that is rapidly metabolized via triose phosphate intermediates to glucose-6-phosphate, lactate and acetyl-CoA thereby further promoting 2 H-enrichment of these metabolites from 2 H 2 O. It is possible that for mice fed a standard chow diet featuring maltose as the main carbohydrate componentwhose digestion yields unlabeled glucose -there could be less complete 2 H incorporation from 2 H 2 O into fatty acids, particularly via NADPH. In cultured cells, N for palmitate was estimated to be 17 (8). This is 55% of the theoretical value and is also substantially lower than that determined for in vivo rat studies. Our data for mice indicate that if glucose was the sole source of DNL in vivo, the value of N would be 24 based on 2 H transfer from exogenous [U-metabolites in vivo compared to in vitro, we anticipate that there would be a greater degree of transfer of 2 H from glucose to fatty acids in vitro, corresponding to a lower N value.
In conclusion, N may vary considerably according to the type of organism as well as the substrates that were utilized for DNL. Our study indicates that transfer of hydrogen from unlabelled glucose to fatty acids via NADPH is an important factor in the non-stoichiometric  n.d. = not detected (signal to noise ratio < 3:1).   include cycling between NADPH and other redox co-factors such as FAD whose hydrogens are exchanged with those of water; generation of NADPH from malate and isocitrate via NADP + -malic enzyme and NADP + -isocitrate dehydrogenase, respectively; and exchange of NADH and NADPH via transhydrogenase. On the right hand side are the two principal pathways that transfer hydrogens to NADPH from nutrient substrates, namely glucose and serine.