Transfer of glucose hydrogens via acetyl-CoA, malonyl-CoA, and NADPH to fatty acids during de novo lipogenesis

Deuterated water (2H2O) is widely used for measuring de novo lipogenesis (DNL). 2H is incorporated into fatty acids via exchange between body water and the hydrogens of acetyl-CoA, malonyl-CoA, and NADPH. Previous studies concluded that these exchanges are incomplete; therefore, fatty acid 2H enrichment requires correcting. In mice, we measured the 2H enrichment of fatty acid positions 2 and 3 and methyl hydrogens from [U-2H7]glucose to determine 2H transfer from glucose to fatty acid via malonyl-CoA, NADPH, and acetyl-CoA, respectively. Positional fatty acid 2H enrichments were compared with 13C enrichment of the same sites from an equivalent amount of [U-13C6]glucose provided alongside the [U-2H7]glucose tracer. Transfer of glucose 2H to fatty acid position 2 and methyl sites was low (2H enrichment of 0.06 ± 0.01 and 0.14 ± 0.01 relative to 13C) indicating extensive exchange at both malonyl- and acetyl-CoA, respectively. Transfer of glucose 2H into fatty acid position 3 was more extensive (0.46 ± 0.04 relative to 13C, P < 10−5 vs. position 2), indicating a more limited exchange of those glucose hydrogens that were transferred via NADPH. However, mice provided with [U-13C6]glucose and 2H2O had equivalent 2H enrichments of fatty acid positions 2 and 3, suggesting that in this setting, NADPH and body water 2H had exchanged extensively. This is explained by contributions of substrates other than exogenous glucose to DNL coupled with their extensive 2H enrichment from 2H2O prior to DNL. Under such conditions, 2H enrichment of fatty acids from 2H2O does not need correction. Deuterated water (2H2O) is widely used for measuring de novo lipogenesis (DNL). 2H is incorporated into fatty acids via exchange between body water and the hydrogens of acetyl-CoA, malonyl-CoA, and NADPH. Previous studies concluded that these exchanges are incomplete; therefore, fatty acid 2H enrichment requires correcting. In mice, we measured the 2H enrichment of fatty acid positions 2 and 3 and methyl hydrogens from [U-2H7]glucose to determine 2H transfer from glucose to fatty acid via malonyl-CoA, NADPH, and acetyl-CoA, respectively. Positional fatty acid 2H enrichments were compared with 13C enrichment of the same sites from an equivalent amount of [U-13C6]glucose provided alongside the [U-2H7]glucose tracer. Transfer of glucose 2H to fatty acid position 2 and methyl sites was low (2H enrichment of 0.06 ± 0.01 and 0.14 ± 0.01 relative to 13C) indicating extensive exchange at both malonyl- and acetyl-CoA, respectively. Transfer of glucose 2H into fatty acid position 3 was more extensive (0.46 ± 0.04 relative to 13C, P < 10−5 vs. position 2), indicating a more limited exchange of those glucose hydrogens that were transferred via NADPH. However, mice provided with [U-13C6]glucose and 2H2O had equivalent 2H enrichments of fatty acid positions 2 and 3, suggesting that in this setting, NADPH and body water 2H had exchanged extensively. This is explained by contributions of substrates other than exogenous glucose to DNL coupled with their extensive 2H enrichment from 2H2O prior to DNL. Under such conditions, 2H enrichment of fatty acids from 2H2O does not need correction. 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.Forcheron F. Cachefo A. Thevenon S. Pinteur C. Beylot M. Mechanisms of the triglyceride- and cholesterol-lowering effect of fenofibrate in hyperlipidemic type 2 diabetic patients.Diabetes. 2002; 51: 3486-3491Crossref PubMed Scopus (115) Google Scholar, 2.Diraison F. Moulin P. Beylot M. Contribution of hepatic de novo lipogenesis and reesterification of plasma non esterified fatty acids to plasma triglyceride synthesis during non-alcoholic fatty liver disease.Diabetes Metab. 2003; 29: 478-485Crossref PubMed Scopus (305) Google Scholar, 3.Saponaro C. Gaggini M. Gastaldelli A. Nonalcoholic fatty liver disease and type 2 diabetes: common pathophysiologic mechanisms.Curr. Diab. Rep. 2015; 15: 607Crossref PubMed Scopus (86) Google Scholar). Fractional DNL rates can be measured from incorporation of deuterated water (2H2O) into fatty acids (4.Diraison F. Pachiaudi C. Beylot M. Measuring lipogenesis and cholesterol synthesis in humans with deuterated water: Use of simple gas chromatographic mass spectrometric techniques.J. Mass Spectrom. 1997; 32: 81-86Crossref PubMed Scopus (106) Google Scholar, 5.Duarte J.A.G. Carvalho F. Pearson M. Horton J.D. Browning J.D. Jones J.G. Burgess S.C. A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.J. Lipid Res. 2014; 55: 2541-2553Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), an inexpensive and simple method that can be applied to humans, animal models, and cell cultures. 2H enrichment of fatty acids from 2H2O is conventionally measured by MS, where all the fatty acid chain hydrogens are considered as a single traceable entity (4.Diraison F. Pachiaudi C. Beylot M. Measuring lipogenesis and cholesterol synthesis in humans with deuterated water: Use of simple gas chromatographic mass spectrometric techniques.J. Mass Spectrom. 1997; 32: 81-86Crossref PubMed Scopus (106) Google Scholar). While this provides amplification of the m+1 signal arising from 2H incorporation, it does not resolve the carbon-bound fatty acid hydrogens according to their metabolic sources (see Fig. 1). It was reported that in rats, about 30% of plasma triglyceride palmitate hydrogens had not exchanged with 2H body water (6.Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 7.Lee W.N. Bassilian S. Ajie H.O. Schoeller D.A. Edmond J. Bergner E.A. Byerley L.O. In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E699-E708Crossref PubMed Google Scholar), while for palmitate derived from cultured cells, this nonexchanged fraction was even higher (8.Lee W.N. Bassilian S. Guo Z. Schoeller D. Edmond J. Bergner E.A. Byerley L.O. Measurement of fractional lipid synthesis using deuterated water (2H2O) and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E372-E383Crossref PubMed Google Scholar). Therefore, for DNL measurement, the 2H enrichment of fatty acids measured by MS needs to be corrected by a predetermined factor related to the number of deuterium atoms that were incorporated per molecule of fatty acid, referred to as N (6.Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (76) Google Scholar). Stoichiometric 2H enrichment of fatty acids from 2H2O 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 2H 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-2H7]glucose and performed 2H NMR analysis of liver triglyceride (5.Duarte J.A.G. Carvalho F. Pearson M. Horton J.D. Browning J.D. Jones J.G. Burgess S.C. A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.J. Lipid Res. 2014; 55: 2541-2553Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 9.Delgado T.C. Pinheiro D. Caldeira M. Castro M. Geraldes C. Lopez-Larrubia P. Cerdan S. Jones J.G. Sources of hepatic triglyceride accumulation during high-fat feeding in the healthy rat.NMR Biomed. 2009; 22: 310-317Crossref PubMed Scopus (46) Google Scholar, 10.Viegas I. Jarak I. Rito J. Carvalho R.A. Meton I. Pardal M.A. Baanante I.V. Jones J.G. Effects of dietary carbohydrate on hepatic de novo lipogenesis in European seabass (Dicentrarchus labrax L.).J. Lipid Res. 2016; 57: 1264-1272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) to determine the extent to which the 2H were transferred into positions 2 and 3 and the terminal methyl hydrogens of hepatic fatty acids: each position reflecting 2H transfer from glucose via malonyl-CoA, NADPH, and acetyl-CoA, respectively. Our data indicate that there was a substantial transfer of glucose hydrogen to newly synthesized 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. [U-2H7]- and [U-13C6]glucose at 98–99% enrichment were obtained from Sigma-Aldrich. [3-2H]glucose at 98% enrichment was obtained from Omicron Biochemicals, South Bend, IN. Animal studies were approved by the University of Coimbra Ethics Committee on Animal Studies (ORBEA) and the Portuguese National Authority for Animal Health (DGAV), approval code 0421/000/000/2013. Adult male C57BL/6J mice were obtained from Charles River Laboratories, Barcelona, Spain, and housed at the University of Coimbra Faculty of Medicine Bioterium, where they were maintained with a 12 h light/12 h dark cycle. Upon delivery to the Bioterium, mice were provided a 2 week interval for acclimation, with free access to water and standard chow. Following acclimation, the drinking water was supplemented with glucose and fructose (15.0 g of each sugar to 100 g of water). In the first set of studies, the glucose was enriched to 20% each with [U-2H7]glucose and [U-13C6]glucose. In a second set of studies, glucose was enriched to 16% each with [3-2H]glucose and [U-13C6]glucose. In the third set of experiments, mice were administered an intraperitoneal injection of 99.9% 2H2O containing 9 mg NaCl per milliliter at a dose of 4 g/100 g body mass at the start of the dark period. For these animals, the drinking water was enriched to 5% with 2H2O and the glucose was enriched to 50% with [U-13C6]glucose but not with 2H. Animals were allowed to feed and drink ad libitum during the entire 12 h dark period and were euthanized by cervical dislocation the following morning. Livers and adipose tissue depots were freeze-clamped and stored at −80°C until further processing for triglyceride extraction and purification. Liver triglyceride was extracted and purified as previously described (10.Viegas I. Jarak I. Rito J. Carvalho R.A. Meton I. Pardal M.A. Baanante I.V. Jones J.G. Effects of dietary carbohydrate on hepatic de novo lipogenesis in European seabass (Dicentrarchus labrax L.).J. Lipid Res. 2016; 57: 1264-1272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Briefly, livers were powdered under and with by was in a for h at and at g for fraction was and was by 4 of water to the fraction and it at for was for at containing the was and under gas in a dark from the lipid were purified with a extraction were with of by of were in of and to the lipid were with a further to transfer the to the were with of in 4 containing were by A of the were on the alongside triglyceride and the was with acid lipid were by were and under gas and stored at until for NMR were in To of a standard enriched to with and in g g and of were and 2H NMR were with an using a with and as previously at were with a 3 and were for each 2H NMR at were obtained with a a an of and a of and were for each were applied to all 2H triglyceride to their relative to the 2H standard were obtained as from a set of liver triglyceride obtained from mice administered with For each a was with the described and by a under the same with the of the and which were set to and respectively. for the 2H in fatty acid position position 3, and methyl hydrogens were and respectively. For 13C isotopomer analysis by 13C triglyceride were in enriched and in 3 NMR 13C NMR were at with an with a 3 were with a an of and a For each were Positional 2H enrichments of triglyceride fatty acids were by analysis of and 2H NMR triglyceride as previously described (5.Duarte J.A.G. Carvalho F. Pearson M. Horton J.D. Browning J.D. Jones J.G. Burgess S.C. A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.J. Lipid Res. 2014; 55: 2541-2553Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 10.Viegas I. Jarak I. Rito J. Carvalho R.A. Meton I. Pardal M.A. Baanante I.V. Jones J.G. Effects of dietary carbohydrate on hepatic de novo lipogenesis in European seabass (Dicentrarchus labrax L.).J. Lipid Res. 2016; 57: 1264-1272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). the methyl and 13C NMR 13C enrichments of fatty acids were from the of derived from to the the 13C (see Fig. the methyl and 13C NMR the 13C enrichment of fatty acids from in the methyl position was as enrichment of fatty acid where and are the and of the of the fatty acid terminal and the 13C enrichment that fatty acid enrichment from via elongation was limited to position and 2 enrichment of 3 was to be equivalent to that of the terminal methyl analysis of the fatty acid and enrichment of the position of fatty acids from was as where and are the and of the and the 13C enrichment enrichment of the fatty acid position 2 was to be to that of position For the fractional of [U-2H7]glucose transfer into and malonyl-CoA relative to that of the enrichment of the terminal methyl and 2 positions were by to for the that there are 2H for 13C in the glucose For the fractional of [U-2H7]glucose transfer into NADPH relative to the of we that 2H were transferred per glucose molecule corresponding to 2H per acetyl-CoA derived from are as ± standard and were by an performed with that were provided with the of [U-2H7]- and [U-13C6]glucose liver that were enriched in both 13C and 2H 1). 2H and 13C NMR of the fatty acid and both of the fatty acid chain Fig. from [U-13C6]glucose was relatively between terminal and indicating that DNL was the for incorporation of glucose into fatty acids with elongation a relatively In enrichment of fatty acid sites from [U-2H7]glucose was as by the of positions 2 and 3 and the terminal methyl and the 2H enrichments from these 1). the fatty acid enrichment from [U-2H7]glucose to that of [U-13C6]glucose provides a of the fractional of the [U-2H7]glucose 2H atoms in a position data that for those fatty acids that were synthesized from exogenous more 2H was transferred into the position 3 hydrogens compared with position 2 the terminal methyl This a exchange of 2H and water hydrogens during the of [U-2H7]glucose to acetyl-CoA and malonyl-CoA compared with hydrogen transfer via and NADPH. of the fatty acid position 2 hydrogens is derived from malonyl-CoA, the other from body Therefore, on the fatty acid position 2 enrichment of we can that the enrichment of the malonyl-CoA was This is to the for the acetyl-CoA by fatty acid 13C and 2H Positional enrichment of fatty acid position was to be equivalent to that of position 20% ± ± enrichment of fatty acid position was to be equivalent to that of position ± enrichment of fatty acid position 3 was to be equivalent to that of the methyl ± ± ± enrichment of fatty acid position was to be equivalent to that of position 16% ± ± ± ± ± acid 13C and 2H enrichments from a of mice provided with glucose enriched to 20% with and [U-2H7]glucose and a second of mice provided with glucose enriched to 16% with and not enrichment of fatty acid position was to be equivalent to that of position enrichment of fatty acid position 3 was to be equivalent to that of the methyl in a enrichment for fatty acid position position 3, and terminal ± < compared with ± < 10−5 compared with ± ± enrichment for fatty acid positions 2 and 3 and from the of mice provided with [U-2H7]- and [U-13C6]glucose and that of fatty position 3 from the of mice provided with and P < compared with P < 10−5 compared with in a acid 13C and 2H enrichments from a of mice provided with glucose enriched to 20% with and [U-2H7]glucose and a second of mice provided with glucose enriched to 16% with and not enrichment for fatty acid positions 2 and 3 and from the of mice provided with [U-2H7]- and [U-13C6]glucose and that of fatty position 3 from the of mice provided with and [U-13C6]glucose. performed an a set of studies where [U-13C6]glucose was by where incorporation of 2H into fatty acids from this occurs via NADPH. into the enrichment 20% for the the fatty acid 13C enrichment were with those of the This that for both studies, exogenous glucose had to the same extent for DNL. a 2H NMR of liver triglyceride from a provided with the In with the metabolic of the 2H fatty acids were enriched in position 3, while enrichment of position 2 was not and the terminal methyl had a 2H the substantial transfer of glucose hydrogens into the position 3 of fatty acid relative to position it be that for mice administered with enrichment of fatty acid position 3 be less than that of position However, as by Fig. the of position 2 and 3 were and there was between the 2H enrichments for each ± and ± for positions 2 and 3, the studies of Beylot and (4.Diraison F. Pachiaudi C. Beylot M. Measuring lipogenesis and cholesterol synthesis in humans with deuterated water: Use of simple gas chromatographic mass spectrometric techniques.J. Mass Spectrom. 1997; 32: 81-86Crossref PubMed Scopus (106) Google Scholar, F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (76) Google Scholar), 2H2O used as a for fractional DNL the it was that 2H incorporation into fatty acids be limited by exchange of body water and hydrogens the incorporation of 2H into these precursors of to In the by fatty acid enrichments corresponding to the transfer of 2H from [U-2H7]glucose via acetyl-CoA, malonyl-CoA, and NADPH, the extent of glucose hydrogen exchange during its to fatty acids via each was of [U-2H7]glucose with a single 2H and a second with 2H in the methyl Therefore, there are of 2H for each this is for lipogenesis via acetyl-CoA, and acetyl-CoA, of the methyl hydrogens exchange with in equivalent of 2H per While methyl hydrogens can extensive exchange with water via F. at of during J. PubMed Scopus (28) Google Scholar), there is that not all in this For in with C. Effects of on and of in the PubMed Scopus Google Scholar) a higher of 2H in compared with at 3 and of with 2H in the methyl position be at rates to those 2H. In the same setting, was less incorporated into the compared with [U-13C6]glucose C. Z. of from glucose in is not by of PubMed Scopus Google Scholar), suggesting that the of 2H in the methyl hydrogens of acetyl-CoA was its to via while there is an exchange for 2H from water into the methyl sites of there be that not this that enriched with 2H be into and acetyl-CoA at rates in to their In we that for the acetyl-CoA that was derived from exogenous of 2H had exchanged for analysis of acetyl-CoA 2H enrichment from 2H2O via (5.Duarte J.A.G. Carvalho F. Pearson M. Horton J.D. Browning J.D. Jones J.G. Burgess S.C. A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.J. Lipid Res. 2014; 55: 2541-2553Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) concluded that exchange between the methyl hydrogens of this and body water was In setting, exogenous glucose a fraction of hepatic DNL C. I. Jones J.G. contributions of exogenous glucose and fructose to de novo fatty acid and synthesis in liver and adipose PubMed Scopus Google Scholar). In the of it is that DNL acetyl-CoA derived from and be enriched to a extent with 2H prior to for DNL. For these we that the hydrogen exchange fraction of the acetyl-CoA is to than to in setting, provided with 2H2O be to 2H enrichments of acetyl-CoA methyl hydrogens and body For [U-2H7]glucose that was to fatty acid via malonyl-CoA, exchange of 2H with was to that for Therefore, enrichment of malonyl-CoA from 2H2O be to that of body 2H that were from glucose to fatty acids via NADPH were more in to those transferred via acetyl-CoA and the first and third hydrogens of glucose are transferred to NADPH, [U-2H7]glucose while [3-2H]glucose In transfer of 2H to fatty acid position 3 from [U-2H7]glucose was to be that from [3-2H]glucose vs. This that under conditions, there was of 2H from glucose position compared with position 3, for by exchange of with and J. of the hydrogen to of glucose in in J. Physiol. PubMed Google Scholar). studies of NADPH enrichment from 2H2O a degree of exchange between the reducing hydrogen and body water than be on Z. J.D. for deuterium of and PubMed Scopus Google Scholar). for this is that the is not the of NADPH, as by Fig. In NADPH can be from by In as as NADPH can be via and In all of these the that the hydrogen that was transferred to had previously exchanged with body water is For NADPH via the the reducing hydrogen from hydrogen 2 of which in from water during the of the is via the to and this hydrogen is transferred to NADPH via of hydrogens are derived from intermediates, such as hydrogens are exchanged with those of In NADPH and water hydrogens be exchanged via Z. J.D. for deuterium of and PubMed Scopus Google Scholar, J. J. J.D. analysis NADPH 2014; PubMed Scopus Google Scholar). and an of of hydrogen exchange at the of NADPH, is that fatty acid 2H enrichment from [U-2H7]glucose via this was relative to [U-13C6]glucose to fatty acid via the of glucose by the was to that of its to fatty acid via malonyl-CoA, the fractional of exchange at the of NADPH from the of fatty acid position However, this does not the that the relative of 2H transferred from glucose to fatty acid via NADPH were higher than those transferred via acetyl-CoA and malonyl-CoA Our of extensive 2H transfer from glucose to fatty acid synthesis via NADPH that this is coupled to the extent that NADPH by the does not with other NADPH This of and NADPH reported in other using and In mice with fatty hepatic measured by a were to be with lipogenesis but were not with lipogenesis but not in J. Physiol. Metab. PubMed Scopus Google Scholar). This that in the NADPH derived from the is for DNL than for such as the of In the cell with D. NADPH in the and of 2014; 55: Full Text Full Text PDF PubMed Scopus Google Scholar) a substantial transfer of 2H from [3-2H]glucose to fatty while fatty acid 2H enrichment via NADPH from was more extensive exchange of exogenous glucose hydrogens with water during its to fatty acids via both and In glucose hydrogens that were transferred to fatty acids via NADPH more limited on these it be that enrichment of the fatty acid position 2 hydrogens from 2H2O from water and be equivalent to those of body while those of position 3 via be less we that positions 2 and 3 were enriched to the same exogenous glucose is a of acetyl-CoA to DNL in it a more substantial to the NADPH for following 2H2O under both and conditions, hepatic enriched with 2H in all positions to extensive between and precursors T.C. Carvalho F. A. Jones J.G. 2H enrichment of hepatic from 2H2O the of dietary fructose to J. Physiol. Metab. PubMed Scopus Google Scholar, S. synthesis in adipose of glucose and 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). Under these conditions, NADPH derived from of enriched with 2H and this be transferred to the fatty acid Under conditions, the number of deuterium atoms incorporated per fatty acid molecule to the theoretical for Therefore, in this needs to be applied to the fatty acid 2H enrichment from This from studies in rats, where N was to be and of and of the theoretical by mass isotopomer analysis for plasma and liver triglyceride (6.Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 7.Lee W.N. Bassilian S. Ajie H.O. Schoeller D.A. Edmond J. Bergner E.A. Byerley L.O. In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E699-E708Crossref PubMed Google Scholar). To N not in the by mass isotopomer but its and higher metabolic it be to be higher compared with the mice in of a sugar that is via to and acetyl-CoA, further 2H enrichment of these from It is that for mice a standard diet as the carbohydrate there be less 2H incorporation from 2H2O into fatty via NADPH. In cultured cells, N for palmitate was to be (8.Lee W.N. Bassilian S. Guo Z. Schoeller D. Edmond J. Bergner E.A. Byerley L.O. Measurement of fractional lipid synthesis using deuterated water (2H2O) and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E372-E383Crossref PubMed Google Scholar). This is of the theoretical and is than that for in vivo Our data for mice indicate that glucose was the of DNL in the of N be on 2H transfer from exogenous [U-2H7]glucose to fatty acids via acetyl-CoA, malonyl-CoA, and there is more for the of glucose 2H by and other transfer of glucose in vivo compared with in we that there be a degree of transfer of 2H from glucose to fatty acids in corresponding to a N In N according to the type of as as the substrates that were for DNL. Our that transfer of hydrogen from glucose to fatty acids via NADPH is an factor in the incorporation of 2H into fatty acids during DNL. For the measurement of DNL with the of 2H enrichment in fatty acid positions 2 and 3 as measured by 2H NMR provides a for the extent to which this with de novo lipogenesis fatty deuterated water the number of deuterium atoms incorporated per fatty acid molecule