The role of hepatic de novo lipogenesis (DNL) in promoting fatty liver disease and hypertriglyceridemia during excessive nutrient intake is becoming firmly established. Certain nutrients such as fructose promote hepatic DNL activity and this has been at least partly attributed to their efficient conversion to the acetyl-CoA precursors of DNL. However, tracer studies indicate a paradoxically low level of fructose incorporation into lipids, which begs the question of what the actual lipogenic acetyl-CoA sources are under these and other conditions. Here, we describe novel approaches for measuring substrate contributions to lipogenic hepatic acetyl-CoA using 13C-tracers and 13C-NMR analysis of lipids and acetyl-CoA probes. We review and address aspects of hepatic intermediary fluxes and acetyl-CoA compartmentation that can confound the relationship between 13C-precursor substrate and lipogenic 13C-acetyl-CoA enrichments and demonstrate novel methodologies that can provide realistic estimates of 13C-enriched substrate contributions to DNL. The most striking realization is that the principal substrate contributors to lipogenic acetyl-CoA have yet to be identified, but they are probably not the so-called “lipogenic substrates” such as fructose. The proposed methods may improve our insight into the nutrient sources of DNL under various feeding and disease states. 1. Overview De novo lipogenesis (DNL) is a constitutive pathway that transforms acetyl-CoA into long-chain fatty acids. In humans, DNL occurs primarily in the liver, [1] while, in rodents and other mammals, adipose tissue can also be important contributors [2, 3]. A widely accepted teleological function of this pathway is the conversion of excess nutrient carbons into an inert and energy-dense triglyceride product that can be stored and mobilized at a later time for energy generation in times of nutrient scarcity. DNL activity is highly regulated both allosterically and transcriptionally such that it normally only operates during conditions of nutrient and energy satiety. For the liver, this represents the absorptive and early postabsorptive feeding phase where there are high portal vein levels of simple sugars and amino acids coupled with increased amounts of insulin. The loss of regulation of hepatic DNL flux secondary to excess nutrient consumption may be an important early event in the development of fatty liver and hypertriglyceridemia, which in turn are harbingers of diabetes and liver disease. Given the steep parallel increases in obesity rates and nonalcoholic fatty liver disease in most
References
[1]
F. Diraison, V. Yankah, D. Letexier, E. Dusserre, P. Jones, and M. Beylot, “Differences in the regulation of adipose tissue and liver lipogenesis by carbohydrates in humans,” Journal of Lipid Research, vol. 44, no. 4, pp. 846–853, 2003.
[2]
D. A. Letexier, C. Pinteur, V. Large, V. Frering, and M. Beylot, “Comparison of lipogenic capacities of human and rat adipose tissues and effects of variation in carbohydrate/lipid diet ratio,” Diabetologia, vol. 46, pp. A216–A217, 2003.
[3]
W. G. Bergen and H. J. Mersmann, “Comparative aspects of lipid metabolism: impact on contemporary research and use of animal models,” Journal of Nutrition, vol. 135, no. 11, pp. 2499–2502, 2005.
[4]
M. A. Solomon, F. M. H. Jeffrey, C. J. Storey, A. D. Sherry, and C. R. Malloy, “Substrate selection early after reperfusion of ischemic regions in the working rabbit heart,” Magnetic Resonance in Medicine, vol. 35, no. 6, pp. 820–826, 1996.
[5]
J. G. Jones, T. H. Le, C. J. Storey, A. D. Sherry, C. R. Malloy, and K. P. Burton, “Effects of different oxidative insults on intermediary metabolism in isolated perfused rat hearts,” Free Radical Biology & Medicine, vol. 20, no. 4, pp. 515–523, 1996.
[6]
F. M. H. Jeffrey, V. Diczku, A. D. Sherry, and C. R. Malloy, “Substrate selection in the isolated working rat heart: effects of reperfusion, afterload, and concentration,” Basic Research in Cardiology, vol. 90, no. 5, pp. 388–396, 1995.
[7]
F. Carvalho, J. Duarte, A. R. Simoes, P. F. Cruz, and J. G. Jones, “Noninvasive measurement of murine hepatic acetyl-CoA 13C-enrichment following overnight feeding with 13C-enriched fructose and glucose,” BioMed Research International, vol. 2013, Article ID 638085, 7 pages, 2013.
[8]
R. R. Gilibili, M. Kandaswamy, K. Sharma, S. Giri, S. Rajagopal, and R. Mullangi, “Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues,” Biomedical Chromatography, vol. 25, no. 12, pp. 1352–1359, 2011.
[9]
A. F. Soares, R. A. Carvalho, F. J. Veiga et al., “Restoration of direct pathway glycogen synthesis flux in the STZ-diabetes rat model by insulin administration,” American Journal of Physiology—Endocrinology and Metabolism, vol. 303, no. 7, pp. E875–E885, 2012.
[10]
T. C. Delgado, D. Pinheiro, M. Caldeira et al., “Sources of hepatic triglyceride accumulation during high-fat feeding in the healthy rat,” NMR in Biomedicine, vol. 22, no. 3, pp. 310–317, 2009.
[11]
C. Barosa, M. Almeida, M. M. Caldeira, F. Gomes, and J. G. Jones, “Contribution of proteolytic and metabolic sources to hepatic glutamine by 2H NMR analysis of urinary phenylacetylglutamine 2H-enrichment from 2H2O,” Metabolic Engineering, vol. 12, no. 1, pp. 53–61, 2010.
[12]
A. M. Silva, F. Martins, J. G. Jones, and R. Carvalho, “2H2O incorporation into hepatic acetyl-CoA and de novo lipogenesis as measured by Krebs cycle-mediated 2H-enrichment of glutamate and glutamine,” Magnetic Resonance in Medicine, vol. 66, no. 6, pp. 1526–1530, 2011.
[13]
F. Carvalho, A. Gon？alves, J. Barra, M. Caldeira, J. Duarte, and J. Jones, “Noninvasive sampling of murine hepatic acetyl-CoA enrichment with p-amino benzoic acid,” Diabetologia, vol. 54, supplement 1, pp. S284–S284, 2011.
[14]
W. N. P. Lee, S. Bassilian, Z. Guo et al., “Measurement of fractional lipid synthesis using deuterated water (2H2O) and mass isotopomer analysis,” The American Journal of Physiology, vol. 266, no. 3, pp. E372–E383, 1994.
[15]
W. N. Lee, S. Bassilian, H. O. Ajie et al., “In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis,” American Journal of Physiology—Endocrinology and Metabolism, vol. 266, no. 5, pp. E699–E708, 1994.
[16]
F. Diraison, C. Pachiaudi, and M. Beylot, “In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated,” Metabolism: Clinical and Experimental, vol. 45, no. 7, pp. 817–821, 1996.
[17]
U. Walter, H. Luthe, F. Gerhart, and H. D. Soeling, “Hydrogen exchange at the β carbon of amino acids during transamination,” European Journal of Biochemistry, vol. 59, no. 2, pp. 395–403, 1975.
[18]
M. K. Hellerstein, M. Christiansen, S. Kaempfer et al., “Measurement of de novo hepatic lipogenesis in humans using stable isotopes,” The Journal of Clinical Investigation, vol. 87, no. 5, pp. 1841–1852, 1991.
[19]
C. des Rosiers, F. David, M. Garneau, and H. Brunengraber, “Nonhomogeneous labeling of liver mitochondrial acetyl-CoA,” The Journal of Biological Chemistry, vol. 266, no. 3, pp. 1574–1578, 1991.
[20]
Y. Zhang, K. C. Agarwal, M. Beylot et al., “Nonhomogeneous labeling of liver extra-mitochondrial acetyl-CoA. Implications for the probing of lipogenic acetyl-CoA via drug acetylation and for the production of acetate by the liver,” The Journal of Biological Chemistry, vol. 269, no. 15, pp. 11025–11029, 1994.
[21]
I. R. Bederman, J. K. Kelleher, D. H. Wasserman, and H. Brunengraber, “Zonation of labeling of lipogenic acetyl-CoA in liver. Implications for measurements of lipogenesis by mass isotopomer analysis,” The FASEB Journal, vol. 15, p. A750, 2001.
[22]
I. R. Bederman, A. E. Reszko, T. Kasumov et al., “Zonation of labeling of lipogenic acetyl-CoA across the liver: implications for studies of lipogenesis by mass isotopomer analysis,” The Journal of Biological Chemistry, vol. 279, no. 41, pp. 43207–43216, 2004.
[23]
K. Ekberg, V. Chandramouli, K. Kumaran, W. C. Schumann, J. Wahren, and B. R. Landau, “Gluconeogenesis and glucuronidation in liver in vivo and the heterogeneity of hepatocyte function,” The Journal of Biological Chemistry, vol. 270, no. 37, pp. 21715–21717, 1995.
[24]
C. R. Malloy, A. D. Sherry, and F. M. H. Jeffrey, “Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers,” American Journal of Physiology—Heart and Circulatory Physiology, vol. 259, no. 3, pp. H987–H995, 1990.
[25]
F. M. H. Jeffrey, A. Rajagopal, C. R. Malloy, and A. Dean Snerry, “C-NMR: a simple yet comprehensive method for analysis of intermediary metabolism,” Trends in Biochemical Sciences, vol. 16, no. 1, pp. 5–10, 1991.
[26]
A. D. Sherry, F. M. H. Jeffrey, and C. R. Malloy, “Analytical solutions for 13C isotopomer analysis of complex metabolic conditions: substrate oxidation, multiple pyruvate cycles, and gluconeogenesis,” Metabolic Engineering, vol. 6, no. 1, pp. 12–24, 2004.
[27]
F. Diraison, C. Pachiaudi, and M. Beylot, “Measuring lipogenesis and cholesterol synthesis in humans with deuterated water: use of simple gas chromatographic mass spectrometric techniques,” Journal of Mass Spectrometry, vol. 32, pp. 81–86, 1997.
[28]
T. C. Delgado, M. Caldeira, M. M. C. A. Castro et al., “Mechanisms of hepatic triglyceride accumulation during high-fat feeding in the healthy rat,” NMR in Biomedicine, vol. 22, pp. 310–317, 2009.
[29]
I. Magnusson, W. C. Schumann, G. E. Bartsch et al., “Noninvasive tracing of Krebs cycle metabolism in liver,” The Journal of Biological Chemistry, vol. 266, no. 11, pp. 6975–6984, 1991.
[30]
J. G. Jones, R. Naidoo, A. D. Sherry, F. M. H. Jeffrey, G. L. Cottam, and C. R. Malloy, “Measurement of gluconeogenesis and pyruvate recycling in the rat liver: a simple analysis of glucose and glutamate isotopomers during metabolism of [1,2,3-13C3]propionate,” The FEBS Letters, vol. 412, no. 1, pp. 131–137, 1997.
[31]
V. Large, H. Brunengraber, M. Odeon, and M. Beylot, “Use of labeling pattern of liver glutamate to calculate rates of citric acid cycle and gluconeogenesis,” American Journal of Physiology—Endocrinology and Metabolism, vol. 272, no. 1, pp. E51–E58, 1997.
[32]
L. Agius and M. Peak, “Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin,” Biochemical Journal, vol. 296, no. 3, pp. 785–796, 1993.
[33]
D. R. Davies, M. Detheux, and E. van Schaftingen, “Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes,” European Journal of Biochemistry, vol. 192, no. 2, pp. 283–289, 1990.
[34]
H. Y. Koo, M. A. Wallig, B. H. Chung, T. Y. Nara, B. H. S. Cho, and M. T. Nakamura, “Dietary fructose induces a wide range of genes with distinct shift in carbohydrate and lipid metabolism in fed and fasted rat liver,” Biochimica et Biophysica Acta: Molecular Basis of Disease, vol. 1782, no. 5, pp. 341–348, 2008.
[35]
T. Kazumi, H. Odaka, T. Hozumi, Y. Ishida, N. Amano, and G. Yoshino, “Effects of dietary fructose or glucose on triglyceride production and lipogenic enzyme activities in the liver of Wistar fatty rats, an animal model of NIDDM,” Endocrine Journal, vol. 44, no. 2, pp. 239–245, 1997.
[36]
K. Lê and L. Tappy, “Metabolic effects of fructose,” Current Opinion in Clinical Nutrition and Metabolic Care, vol. 9, no. 4, pp. 469–475, 2006.
[37]
E. J. Parks, L. E. Skokan, M. T. Timlin, and C. S. Dingfelder, “Dietary sugars stimulate fatty acid synthesis in adults,” Journal of Nutrition, vol. 138, no. 6, pp. 1039–1046, 2008.
[38]
S. Z. Sun and M. W. Empie, “Fructose metabolism in humans: what isotopic tracer studies tell us,” Nutrition and Metabolism, vol. 9, article 89, 2012.
[39]
M. F. F. Chong, B. A. Fielding, and K. N. Frayn, “Mechanisms for the acute effect of fructose on postprandial lipemia,” The American Journal of Clinical Nutrition, vol. 85, no. 6, pp. 1511–1520, 2010.
[40]
E. F. Murphy, P. D. Cotter, S. Healy et al., “Composition and energy harvesting capacity of the gut microbiota: relationship to diet, obesity and time in mouse models,” Gut, vol. 59, no. 12, pp. 1635–1642, 2010.
[41]
J. M. W. Wong, R. de Souza, C. W. C. Kendall, A. Emam, and D. J. A. Jenkins, “Colonic health: fermentation and short chain fatty acids,” Journal of Clinical Gastroenterology, vol. 40, no. 3, pp. 235–243, 2006.
