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• W2007691161 abstract "The primary products of de novo lipogenesis (DNL) are saturated fatty acids, which confer adverse cellular effects. Human adipocytes differentiated with no exogenous fat accumulated triacylglycerol (TG) in lipid droplets and differentiated normally. TG composition showed the products of DNL (saturated fatty acids from 12:0 to 18:0) together with unsaturated fatty acids (particularly 16:1n-7 and 18:1n-9) produced by elongation/desaturation. There was parallel upregulation of expression of genes involved in DNL and in fatty acid elongation and desaturation, suggesting coordinated control of expression. Enzyme products (desaturation ratios, elongation ratios, and total pathway flux) were also correlated with mRNA levels. We used 13C-labeled substrates to study the pathway of DNL. Glucose (5 mM or 17.5 mM in the medium) provided less than half the carbon used for DNL (42% and 47%, respectively). Glutamine (2 mM) provided 9-10%, depending upon glucose concentration. In contrast, glucose provided most (72%) of the carbon of TG-glycerol. Pathway analysis using mass isotopomer distribution analysis (MIDA) revealed that the pathway for conversion of glucose to palmitate is complex. DNL in human fat cells is tightly coupled with further modification of fatty acids to produce a range of saturated and unsaturated fatty acids consistent with normal maturation. The primary products of de novo lipogenesis (DNL) are saturated fatty acids, which confer adverse cellular effects. Human adipocytes differentiated with no exogenous fat accumulated triacylglycerol (TG) in lipid droplets and differentiated normally. TG composition showed the products of DNL (saturated fatty acids from 12:0 to 18:0) together with unsaturated fatty acids (particularly 16:1n-7 and 18:1n-9) produced by elongation/desaturation. There was parallel upregulation of expression of genes involved in DNL and in fatty acid elongation and desaturation, suggesting coordinated control of expression. Enzyme products (desaturation ratios, elongation ratios, and total pathway flux) were also correlated with mRNA levels. We used 13C-labeled substrates to study the pathway of DNL. Glucose (5 mM or 17.5 mM in the medium) provided less than half the carbon used for DNL (42% and 47%, respectively). Glutamine (2 mM) provided 9-10%, depending upon glucose concentration. In contrast, glucose provided most (72%) of the carbon of TG-glycerol. Pathway analysis using mass isotopomer distribution analysis (MIDA) revealed that the pathway for conversion of glucose to palmitate is complex. DNL in human fat cells is tightly coupled with further modification of fatty acids to produce a range of saturated and unsaturated fatty acids consistent with normal maturation. The pathway of de novo lipogenesis (DNL) was, until recently, believed to be virtually nonexistent in the human adipocyte (1Shrago E. Spennetta T. Gordon E. Fatty acid synthesis in human adipose tissue.J. Biol. Chem. 1969; 244: 2761-2766Abstract Full Text PDF PubMed Google Scholar, 2Patel M.S. Owen O.E. Goldman L.I. Hanson R.W. Fatty acid synthesis by human adipose tissue.Metabolism. 1975; 24: 161-173Abstract Full Text PDF PubMed Scopus (51) Google Scholar, 3Galton D.J. Lipogenesis in human adipose tissue.J. Lipid Res. 1968; 9: 19-26Abstract Full Text PDF PubMed Google Scholar). However, it is becoming increasingly clear that human adipocytes are capable of synthesis of fatty acids and triacylglycerols (TG) from nonlipid precursors. The expression of key enzymes of DNL, including acetyl-CoA carboxylase-A (ACACA) and fatty acid synthase (FAS), has been demonstrated in human adipose tissue (4Letexier D. Pinteur C. Large V. Fréring V. Beylot M. Comparison of the expression and activity of the lipogenic pathway in human and rat adipose tissue.J. Lipid Res. 2003; 44: 2127-2134Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 5Roberts R. Hodson L. Dennis A.L. Neville M.J. Humphreys S.M. Harnden K.E. Micklem K.J. Frayn K.N. Markers of de novo lipogenesis in adipose tissue: associations with small adipocytes and insulin sensitivity in humans.Diabetologia. 2009; 52: 882-890Crossref PubMed Scopus (197) Google Scholar). Functionally, around 20% of palmitic acid in adipocyte TG arises from DNL, and although hepatic DNL could account for some of this (exported to the adipocyte in very low density lipoprotein TG), there is an excess that appears to have arisen in the adipose tissue (6Strawford A. Antelo F. Christiansen M. Hellerstein M.K. Adipose tissue triglyceride turnover, de novo lipogenesis, and cell proliferation in humans measured with 2H2O.Am. J. Physiol. Endocrinol. Metab. 2004; 286: E577-E588Crossref PubMed Scopus (270) Google Scholar). During hypercaloric, high-carbohydrate feeding, whole body DNL (measured by indirect calorimetry) increases considerably (7Pasquet P. Brigant L. Froment A. Koppert G.A. Bard D. de Garine I. Apfelbaum M. Massive overfeeding and energy balance in men: the Guru Walla model.Am. J. Clin. Nutr. 1992; 56: 483-490Crossref PubMed Scopus (76) Google Scholar) and exceeds hepatic DNL (measured with tracers); the remainder may occur in adipose tissue (8Aarsland A. Chinkes D. Wolfe R.R. Hepatic and whole-body fat synthesis in humans during carbohydrate overfeeding.Am. J. Clin. Nutr. 1997; 65: 1774-1782Crossref PubMed Scopus (156) Google Scholar). The expression of lipogenic enzymes, which are greater in the adipose tissue of lean people (9Diraison F. Dusserre E. Vidal H. Sothier M. Beylot M. Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity.Am. J. Physiol. Endocrinol. Metab. 2002; 282: E46-E51Crossref PubMed Google Scholar, 10Minehira K. Vega N. Vidal H. Acheson K. Tappy L. Effect of carbohydrate overfeeding on whole body macronutrient metabolism and expression of lipogenic enzymes in adipose tissue of lean and overweight humans.Int. J. Obes. 2004; 28: 1291-1298Crossref PubMed Scopus (77) Google Scholar), specifically, those with smaller adipocytes (5Roberts R. Hodson L. Dennis A.L. Neville M.J. Humphreys S.M. Harnden K.E. Micklem K.J. Frayn K.N. Markers of de novo lipogenesis in adipose tissue: associations with small adipocytes and insulin sensitivity in humans.Diabetologia. 2009; 52: 882-890Crossref PubMed Scopus (197) Google Scholar), relates inversely to obesity. This finding is paralleled by accumulation of products of DNL, especially stearic acid (18:0) (5Roberts R. Hodson L. Dennis A.L. Neville M.J. Humphreys S.M. Harnden K.E. Micklem K.J. Frayn K.N. Markers of de novo lipogenesis in adipose tissue: associations with small adipocytes and insulin sensitivity in humans.Diabetologia. 2009; 52: 882-890Crossref PubMed Scopus (197) Google Scholar). This observation might suggest that small adipocytes use DNL to begin the process of lipid accumulation, with pathways for uptake of extracellular fatty acids becoming more important as the cells develop. Indeed, the lipogenic capacity of fetal human fat cells is high as the cells develop into mature adipocytes (11Dunlop M. Court J.M. Lipogenesis in developing human adipose tissue.Early Hum. Dev. 1978; 2: 123-130Crossref PubMed Scopus (24) Google Scholar). There is further evidence from the study of human adipocyte differentiation in culture. Human preadipocytes differentiate and accumulate lipid droplets in the complete absence of an exogenous fat source (12Hauner H. Skurk T. Wabitsch M. Cultures of human adipose precursor cells.Methods Mol. Biol. 2001; 155: 239-247PubMed Google Scholar). All of their accumulated fat should, therefore, be the product of DNL. The primary product of DNL is palmitic acid (16:0), a saturated fatty acid. There is considerable evidence for adverse effects of excessive saturated fatty acid enrichment of cells (13Busch A.K. Gurisik E. Cordery D.V. Sudlow M. Denyer G.S. Laybutt D.R. Hughes W.E. Biden T.J. Increased fatty acid desaturation and enhanced expression of stearoyl coenzyme A desaturase protects pancreatic β-cells from lipoapoptosis.Diabetes. 2005; 54: 2917-2924Crossref PubMed Scopus (147) Google Scholar, 14Moffitt J.H. Fielding B.A. Evershed R. Berstan R. Currie J.M. Clark A. Adverse physicochemical properties of tripalmitin in beta cells lead to morphological changes and lipotoxicity in vitro.Diabetologia. 2005; 48: 1819-1829Crossref PubMed Scopus (97) Google Scholar, 15Borradaile N.M. Han X. Harp J.D. Gale S.E. Ory D.S. Schaffer J.E. Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death.J. Lipid Res. 2006; 47: 2726-2737Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). Therefore, adipocyte DNL might be expected to be associated with an adverse metabolic profile. It was surprising to discover that smaller cells that are associated with insulin sensitivity in vivo display more signs of DNL (i.e., increased expression of lipogenic enzymes and increased content of saturated fatty acids, especially myristic acid 14:0 and stearic acid) than do larger cells that are associated with insulin resistance in vivo (5Roberts R. Hodson L. Dennis A.L. Neville M.J. Humphreys S.M. Harnden K.E. Micklem K.J. Frayn K.N. Markers of de novo lipogenesis in adipose tissue: associations with small adipocytes and insulin sensitivity in humans.Diabetologia. 2009; 52: 882-890Crossref PubMed Scopus (197) Google Scholar). However, the palmitic acid content of adipose tissue TG does not relate to any of these measurements (5Roberts R. Hodson L. Dennis A.L. Neville M.J. Humphreys S.M. Harnden K.E. Micklem K.J. Frayn K.N. Markers of de novo lipogenesis in adipose tissue: associations with small adipocytes and insulin sensitivity in humans.Diabetologia. 2009; 52: 882-890Crossref PubMed Scopus (197) Google Scholar). These observations suggested that the differentiating adipocyte might defend itself against adverse accumulation of saturated fatty acids produced from DNL by close linkage of DNL with further modification of fatty acids. There is evidence that hepatic DNL is regulated in parallel with elongation of fatty acids and their desaturation by the enzyme stearoyl-CoA desaturase (SCD, or delta-9 desaturase) (16Chong M.F. Hodson L. Bickerton A.S. Roberts R. Neville M. Karpe F. Frayn K.N. Fielding B.A. Parallel activation of de novo lipogenesis and stearoyl-CoA desaturase activity after 3 d of high-carbohydrate feeding.Am. J. Clin. Nutr. 2008; 87: 817-823Crossref PubMed Scopus (164) Google Scholar, 17Oosterveer M.H. van Dijk T.H. Tietge U.J. Boer T. Havinga R. Stellaard F. Groen A.K. Kuipers F. Reijngoud D-J. High fat feeding induces hepatic fatty acid elongation in mice.PLoS O NE. 2009; 4: e6066Crossref PubMed Scopus (117) Google Scholar). We recently showed that differentiating human adipocytes protect themselves against cytotoxic effects of exogenous palmitate by upregulation of pathways for oleate synthesis (DNL, elongation and desaturation) (18Collins J.M. Neville M.J. Hoppa M.B. Frayn K.N. De novo lipogenesis and stearoyl-CoA desaturase are coordinately regulated in the human adipocyte and protect against palmitate-induced cell injury.J. Biol. Chem. 2010; 285: 6044-6052Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). However, the coordinate regulation of these processes has not previously been shown in the differentiating adipocyte dependent solely on DNL-derived fatty acids for lipid accumulation. Therefore, we investigated the induction of DNL during human adipocyte differentiation. Our hypothesis was that DNL could provide all the fatty acids necessary for normal differentiation and maturation if pathways for elongation and desaturation were induced in parallel. We also examined the pathways for DNL in the differentiating human adipocyte, a subject on which there is very little information. Although our starting hypothesis was that exogenous glucose would be the major precursor, we discovered that a large proportion of the fatty acids are synthesized from other sources. We explored that observation and details of the pathways involved. Subcutaneous adipose tissue biopsies were obtained by needle aspiration using a 12 gauge needle. Tissue donors consisted of 30 males and 39 females, with a median age of 41 years (ranging from 26 to 53 years), and with a median body mass index (BMI) of 26.3 kg/m2 (ranging from 16.7 to 40.1 kg/m2). The taking of human adipose tissue samples was approved by the Oxfordshire Clinical Research Ethics Committee, and all subjects gave written, informed consent. Preadipocytes were isolated from subcutaneous adipose tissue and grown in medium consisting of Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham's (v/v, 1:1), 10% fetal calf serum (Invitrogen), 1 µl/100 ml fibroblast growth factor and 100 units/ml penicillin as described previously (18Collins J.M. Neville M.J. Hoppa M.B. Frayn K.N. De novo lipogenesis and stearoyl-CoA desaturase are coordinately regulated in the human adipocyte and protect against palmitate-induced cell injury.J. Biol. Chem. 2010; 285: 6044-6052Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Fully confluent preadipocytes (day 0) were then differentiated for 14 days using a differentiation medium based on that of Hauner et al. (12Hauner H. Skurk T. Wabitsch M. Cultures of human adipose precursor cells.Methods Mol. Biol. 2001; 155: 239-247PubMed Google Scholar), consisting of Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (v/v, 1:1) containing 2 mM glutamine, 17 µM pantothenate, 100 nM human insulin, 1 nM triiodo-L-thyronine, 33 µM biotin, 10 µg/ml transferrin, 1 µM dexamethasone, and 1 ml/l gentamycin. 3-Isobutyl-1-methylxanthine (250 µM) and troglitazone (2 µM) were added for the first 3 days. As discussed previously (18Collins J.M. Neville M.J. Hoppa M.B. Frayn K.N. De novo lipogenesis and stearoyl-CoA desaturase are coordinately regulated in the human adipocyte and protect against palmitate-induced cell injury.J. Biol. Chem. 2010; 285: 6044-6052Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), the cells are differentiated without a source of exogenous fatty acids. Therefore all lipids present at day 14 were either present at the start of differentiation or arose through DNL. Total adipocyte protein per T25 flask was determined with the Bradford method (19Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar) using Coomassie Blue G (Sigma-Aldrich, Gillingham, UK) made up according to the manufacturer's instructions. Bovine serum albumin (Sigma-Aldrich) was included as a standard. Fatty acid methyl esters (FAME) of adipocyte TGs and phospholipids (PL) were prepared and analyzed by gas chromatography (GC) as described previously (18Collins J.M. Neville M.J. Hoppa M.B. Frayn K.N. De novo lipogenesis and stearoyl-CoA desaturase are coordinately regulated in the human adipocyte and protect against palmitate-induced cell injury.J. Biol. Chem. 2010; 285: 6044-6052Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The fatty acid concentrations were calculated relative to the internal standard, and the results were expressed either as micrograms of fatty acid per 106 cells or as a mole percentage. Flux of fatty acids through the enzymes SCD and elongation of long chain fatty acids 6 (ELOVL6) was estimated as the sum of all the fatty acid products of the enzymes (18Collins J.M. Neville M.J. Hoppa M.B. Frayn K.N. De novo lipogenesis and stearoyl-CoA desaturase are coordinately regulated in the human adipocyte and protect against palmitate-induced cell injury.J. Biol. Chem. 2010; 285: 6044-6052Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and was expressed as micromoles of fatty acid over 14 days per 106 cells. For SCD, this was the sum of 16:1 n-7, 18:1 n-7, and 18:1 n-9, and for ELOVL6, the sum of 18:0 and 18:1 n-7. Isotopically (13C)-labeled substrates (CK Gas, Cambridgeshire, UK) were added to the culture medium throughout differentiation to determine the precursors for DNL. In separate experiments, the following substrates were used: 1 mM [1-13C]acetate, D-[U-13C]glucose, 0.5 mM [U-13C]pyruvate, and 2 mM [U-13C]glutamine. For all except [1-13C]acetate, two glucose concentrations were studied, low (5 mM) and high (17.5 mM). Again, for all except [1-13C]acetate, these labeled substrates replaced the corresponding unlabeled substrate normally present in the medium. After lipid extraction and separation of the TG fraction, FAMEs were analyzed using gas chromatography-mass spectrometry (GC-MS) (18Collins J.M. Neville M.J. Hoppa M.B. Frayn K.N. De novo lipogenesis and stearoyl-CoA desaturase are coordinately regulated in the human adipocyte and protect against palmitate-induced cell injury.J. Biol. Chem. 2010; 285: 6044-6052Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). TG glycerol was extracted from the aqueous phase of the fatty acid derivitization, and tertiary-butyldimethylsilyl glycerol derivatives were produced as previously described (20Adiels M. Larsson T. Sutton P. Taskinen M-R. Borén J. Fielding B.A. Optimization of N-methyl-N-[tert-butyldimethylsilyl]trifluoroacetamide as a derivatization agent for determining isotopic enrichment of glycerol in very-low density lipoproteins.Rapid Commun. Mass Spectrom. 2010; 24: 586-592Crossref PubMed Scopus (9) Google Scholar). Glycerol was measured using a GC-MS equipped with a DB-Wax 30 m capillary column (i.d. 0.25 mm, film thickness 0.25 µm; Agilent). The sample (1 µl) was injected onto the column at an initial oven temperature of 110°C. The oven temperature was ramped at 10°C/min to 210°C and then at 20°C/min to 310°C. It was held at this temperature for 3 min for a total of 18 min. Injector and interface were 320°C, and gas flow was 1.1 ml/min. The sample was injected at split ratio of 50:1. The major peak corresponds to a fragmentation where a butyl group is removed and results in a molecule with a mass to charge ratio (m/z) of 377.3; thus, the peaks of m/z 377.3 and m/z 382.3 were measured using selected ion monitoring. Glycerol was corrected for natural enrichment using the isotope pattern obtained from Sheffield ChemPuter (http://winter.group.shef.ac.uk/chemputer/). GC-MS analysis produced a mass spectrum for each fatty acid and the relative abundance of each mass isotopomer. For each fatty acid, the native, unlabeled isotopomer is denoted M+0; e.g., methyl-palmitate M+0 has a mass of 270.3, whereas the isotopomer for [13C1]methyl-palmitate (M+1) has a mass of 271.3, etc. Relative abundance values were corrected for the natural abundance of 13C, which was obtained from measurements made in cells with no added labeled substrate. Data were analyzed in two ways: quantitative mass spectral analysis (QMSA) and mass isotopomer distribution analysis (MIDA). QMSA was based on the method described by Tayek and Katz (21Tayek J.A. Katz J. Glucose production, recycling, and gluconeogenesis in normals and diabetics: a mass isotopomer [U-13C]glucose study.Am. J. Physiol. 1996; 270: E709-E717PubMed Google Scholar). In essence, we calculated the fraction of all carbon atoms in the product (e.g., palmitic acid) that were 13C. As the substrates [U13C]glucose and [U13C]glutamine were uniformly labeled, this represented the fraction of the product formed from the substrate in question. The same calculation was applied to the TG-glycerol moiety. The evaluation of DNL through MIDA assumes that the fatty acids are constructed as polymers of the 2-carbon building block acetyl-CoA. If the 13C-enrichment of the acetyl-CoA pool is known, then the pattern of labeled molecules (mass isotopomers) produced can be predicted by the binomial theorem (22Hellerstein M.K. Christiansen M. Kaempfer S. Kletke S. Wu K. Reid J.S. Mulligan K. Hellerstein N.S. Shackleton C.H.L. Measurement of de novo lipogenesis in humans using stable isotopes.J. Clin. Invest. 1991; 87: 1841-1852Crossref PubMed Scopus (296) Google Scholar). Our data did not fit this simple model (see Results); therefore, more sophisticated modeling was attempted (see the supplementary data). RNA extracted from adipocytes was used to synthesize cDNA for real-time polymerase chain reaction (PCR) analysis as previously described using 500 ng RNA (18Collins J.M. Neville M.J. Hoppa M.B. Frayn K.N. De novo lipogenesis and stearoyl-CoA desaturase are coordinately regulated in the human adipocyte and protect against palmitate-induced cell injury.J. Biol. Chem. 2010; 285: 6044-6052Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Target genes were as follows: ACACA, ACLY, ADIPOQ, CEBPA, DGAT2, ELOVL6, FABP4, FASN, G6PD, PCK1, PPARG, SCD, and SREBF1 (assay IDs Hs00167385_m1, Hs00153764_m1, Hs00605917_m1, Hs00269972_s1, Hs00261438, Hs00225412_m1, Hs00609791m1, Hs00188012_m1, Hs00166169_m1, Hs00159918_m1, Hs00234592_m1, Hs00748952_s1, and Hs00231674_m1, respectively). Normalized mRNA expression was calculated for each target gene using the Δ/ΔCT relative quantitation calculation as previously described (23Risérus U. Tan G.D. Fielding B.A. Neville M.J. Currie J. Savage D.B. Chatterjee V.K. Frayn K.N. O'Rahilly S. Karpe F. Rosiglitazone increases indexes of stearoyl-CoA desaturase activity in humans: link to insulin sensitization and the role of dominant-negative mutation in peroxisome proliferator-activated receptor-gamma.Diabetes. 2005; 54: 1379-1384Crossref PubMed Scopus (101) Google Scholar, 24Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25424) Google Scholar). In brief, the ΔCT transformation of all samples for each transcript was first calculated as ΔCT=E[minCT-sampleCT], where E equals the efficiency of the qRT-PCR reaction as calculated from the slope of a standard curve generated from a serial dilution of a pool of cDNA from all samples [E = (10[-1/slope])]. The Δ/ΔCT for each target gene was calculated as the target gene ΔCT divided by the ΔCT for the stable endogenous control PPIA (cyclophilin; assay ID Hs99999906_m1) (25Neville M.J. Collins J.M. Gloyn A.L. McCarthy M.I. Karpe F. Comprehensive human adipose tissue mRNA and microRNA endogenous control selection for quantitative real-time-PCR normalization.Obesity (Silver Spring). 2011; 19: 888-892Crossref PubMed Scopus (99) Google Scholar). All measurements were made in triplicate. Differences occurring over time were statistically analyzed using repeated-measures (ANOVA). Values were log-transformed where appropriate to achieve normality. Differences between conditions (e.g., low and high glucose concentrations) were assessed using a Student's paired t-test. Correlations were assessed using Spearman's rank correlation coefficient, rs. All statistical analyses were carried out using SPSS (version 15). TG accumulated in cells during the differentiation process (Figs. 1 and 2A). The PL content tended to increase during the first week, and then it was constant (Fig. 2B). The protein content (expressed per 106 cells) rose slightly from preadipocyte to day 7, but then it was relatively constant (mean values for preadipocytes and for days 7, 10, and 14, respectively, were 198, 229, 238, and 225 µg (n = 3, P = 0.82, ANOVA).Fig. 2TG and PL content of cells during differentiation and fatty acid composition. A: TG content during differentiation; n = 5, P = 0.06 for effect of time. B: PL content during differentiation; n = 5, P = 0.16. C: TG fatty acid composition, n = 5 for days 0-10, n = 74 at day 14, main effects of day (P = 0.04), fatty acid (P < 0.001), and day × fatty acid interaction (P < 0.001). D: PL fatty acid composition, n = 5, day × fatty acid interaction, P < 0.001. All statistics by repeated measures ANOVA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The fatty acid composition of TG changed during differentiation. Stearic acid (18:0) predominated at early times, but it decreased, so that 16:0 (palmitic acid) became the major fatty acid by day 14 (Fig. 2C). The proportion of the essential fatty acid 18:2n-6 (linoleic acid) fell during this period. However, because the total TG content increased, the amount (µmol) of 18:2n-6 in TG changed much less, from 523 ± 83 µmol/106 cells in preadipocytes to 446 ± 55 µmol/106 cells at day 14 (P = 0.36, ANOVA). Therefore, the major fatty acids in TG at day 14 were those expected from the coordinate operation of the DNL and the elongation and desaturation pathways: 16:0, 16:1n-7 (palmitoleic acid), 18:0, 18:1n-9 (oleic acid), and 18:1n-7 (cis-vaccenic or asclepic acid, the elongation product of 16:1n-7). Smaller amounts of 12:0, 14:0 (Fig. 2C), and 14:1n-5 (the desaturation product of 14:0, not shown, mol% 0.20 ± 0.12) were also found. The 16:0/18:2n-6 ratio was calculated as an index of DNL (dilution of essential fatty acids by those synthesized de novo) (16Chong M.F. Hodson L. Bickerton A.S. Roberts R. Neville M. Karpe F. Frayn K.N. Fielding B.A. Parallel activation of de novo lipogenesis and stearoyl-CoA desaturase activity after 3 d of high-carbohydrate feeding.Am. J. Clin. Nutr. 2008; 87: 817-823Crossref PubMed Scopus (164) Google Scholar, 26Hudgins L.C. Hellerstein M. Seidman C. Neese R. Diakun J. Hirsch J. Human fatty acid synthesis is stimulated by a eucaloric low fat, high carbohydrate diet.J. Clin. Invest. 1996; 97: 2081-2091Crossref PubMed Scopus (284) Google Scholar). It rose from 1.5 ± 0.2 at day 0 to 39.9 ± 27.8 at day 14 (P = 0.04, ANOVA). The PL fatty acid composition changed less (Fig. 2D). There was a progressive increase in the proportion of 16:0, whereas that of 18:2n-6 decreased. Again, the 16:0/18:2n-6 ratio rose from 1.3 ± 0.1 at day 0 to 3.7 ± 1.2 at day 14 (P = 0.01, ANOVA). The changes in TG amount and composition were mirrored by the mRNA expression of the genes involved in DNL, elongation, and desaturation, as well as “classical” markers of differentiation, including CEBPA, PPARG, LPL, adiponectin, and leptin (Fig. 3). At day 14, the cells were expressing genes for fatty acid synthesis (ACACA, FASN, SCD, ELOVL6) as well as TG synthesis (e.g., DGAT2) (supplementary Table I). Genes involved in the supply of cytosolic acetyl-CoA for DNL (ACLY) and in the supply of NADPH (ME1, G6PD) were also expressed, as was the key glyceroneogenic gene PCK1 (PEPCK-cytosolic). Fatty acid ratios reflecting the action of SCD, 16:1n-7/16:0 and 18:1n-9/18:0, were highly correlated across all cell cultures studied (Fig. 4A). Each of these ratios also correlated with the mRNA expression of SCD (rs= 0.37, P = 0.01; rs= 0.67, P < 0.001, respectively). Fatty acid ratios reflecting the action of ELOVL6, 18:0/16:0 and 18:1n-7/16:1n-7, were also correlated (Fig. 4B). However, ELOVL6 mRNA did not correlate with these fatty acid ratios, probably because one reaction removes reactants from another. We also calculated flux through each of these reactions, based on total accumulation of their products in TG over 14 days. In each case, flux correlated with mRNA expression of the respective enzyme (Fig. 4C, D). The various mRNAs measured also tended to correlate highly with one another (Table 1), implying a strong element of coordinate control of gene expression in these pathways. Interestingly, one exception was SREBF1, in which mRNA did not correlate significantly with that of any other gene studied.TABLE 1Relationships among mRNA levels for genes involved in differentiation, DNL, and fatty acid modificationPPARGSREBF1ACLYG6PDACACAFASNSCDAELOVL6DGAT2CEBPArs0.9150.0270.8800.5980.7670.9030.9160.6430.922p0.0000.8720.0000.0000.0000.0000.0000.0860.000n39393930393939834PPARGrs0.0490.8540.6060.7090.8600.9080.8250.895p0.7200.0000.0000.0000.0000.0000.0000.000n5555375553551543SREBF1rs0.0830.099−0.0290.1140.0790.214−0.059p0.5460.5610.8350.4150.5650.4430.705n55375553551543ACLYrs0.6560.8580.9220.9180.7210.894p0.0000.0000.0000.0000.0020.000n375553551543G6PDrs0.6580.6000.4980.0320.622p0.0000.0000.0020.9090.000n3737371530ACACArs0.8150.7720.5860.846p0.0000.0000.0220.000n53551543FASNrs0.9180.8290.911p0.0000.0000.000n531541SCDrs0.8390.924p0.0000.000n1543ELOVL6rs0.905p0.002n8All measurements were made after 14 days of differentiation. Open table in a new tab All measurements were made after 14 days of differentiation. Next we investigated the pathway of DNL by differentiating cells in the presence of 1 mM [1-13C]acetate. The mass isotopomer spectrum of TG-palmitate from the differentiated cells at day 14 showed relatively consistent labeling from M+1 to M+5, with smaller amounts of higher labeling and none detectable above M+9 (Fig. 5A). (As noted in Experimental Procedures, all fatty acid mass abundance values were corrected for natural abundance by subtraction of results from cells with no added labeled substrate.) Similar patterns of labeling were seen in other TG-fatty acids, 14:0, 16:1n-7, 18:0, 18:1n-7 and 18:1n-9, confirming their origin from DNL. The molar abundance of native (unlabeled) TG-palmitate (M) was 29.8%, implying that the majority of the TG-palmitate molecules incorporated some labeled acetate. QMSA (see Experimental Procedures) showed that 16.6% of the palmitate carbon arose from acetate-13C in the medium (this would equate to 33.2% of palmitate carbon arising from acetate in the medium, since the acetate was labeled on one carbon) (Table 2). This implies considerable dilution of the labeled acetyl-CoA pool with acetyl-CoA coming from unlabeled substrates (e.g., glucose).TABLE 2Percentage of carbon formed from labeled substrate in mediumTG-PalmitateTG-GlycerolSubstrate5 mM Glucos" @default.
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