Differences in the lipid patterns during maturation of 3T3-L1 adipocytes investigated by thin-layer chromatography, gas chromatography, and mass spectrometric approaches

  • Yulia PopkovaEmail author
  • Dirk Dannenberger
  • Jürgen Schiller
  • Kathrin M. Engel
Research Paper
Part of the following topical collections:
  1. Current Progress in Lipidomics


Populations of industrialized countries have registered a dramatically increasing prevalence in obesity for many years. Despite continuous research, mechanisms involved in the storage and utilization of chemical energy in adipocytes are still under investigation. Adipocytes have the task to store excessive energy in the form of triacylglycerols (TG) and it is already well-known that the fatty acyl composition of TG is largely determined by the composition of the diet. In contrast to TG, the composition of adipocyte phospholipids was less comprehensively investigated. In this study, the compositions of the most abundant phospholipid classes of 3T3-L1 undifferentiated (preadipocytes) and differentiated cells (adipocytes) were determined. The lipid fractions were isolated by normal phase high-performance thin-layer chromatography and subsequently analyzed by electrospray ionization mass spectrometry. Additionally, the fatty acyl (FA) compositions were determined by gas chromatography. The positions of the FA residues were additionally confirmed by phospholipase A2 digestion. The advantages and disadvantages of the different analytical approaches will be discussed. It will be shown that undifferentiated 3T3-L1 and mature adipocytes differ extremely regarding their compositions. This goes along with an increase in odd-chain fatty acids.

Graphical abstract


3T3-L1 adipocytes Odd-chain fatty acids ESI mass spectrometry Thin-layer chromatography Gas chromatography Phospholipase A2 digestion 



We thank Prof. Dr. Ines Liebscher, Dr. Doreen Thor, and Tomáš Suchý for providing 3T3-L1 cells and the relevant microscopic images.

Funding information

This study was supported by the German Research Council (SFB 1052/Z3 (Project Number 209933838)). We are particularly indebted to the MERCK Company for the continuous support.

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_2243_MOESM1_ESM.pdf (257 kb)
ESM 1 (PDF 257 kb)


  1. 1.
    Green H, Kehinde O. An established preadipose cell line and its differentiation in culture II. Cell: Factors affecting the adipose conversion; 1975. .CrossRefGoogle Scholar
  2. 2.
    Zhang X, Heckmann BL, Liu J. Studying lipolysis in adipocytes by combining siRNA knockdown and adenovirus-mediated overexpression approaches. Methods Cell Biol. 2013. .Google Scholar
  3. 3.
    Su X, Han X, Yang J, Mancuso DJ, Chen J, Bickel PE, et al. Sequential ordered fatty acid α oxidation and Δ9 desaturation are major determinants of lipid storage and utilization in differentiating adipocytes. Biochemistry. 2004. .CrossRefGoogle Scholar
  4. 4.
    Park BH, Kim DS, Won GW, Jeon HJ, Oh B-C, Lee Y, et al. Mammalian ste20-like kinase and SAV1 promote 3T3-L1 adipocyte differentiation by activation of PPARγ. PLoS One. 2012. .CrossRefGoogle Scholar
  5. 5.
    Guo L, Zhou D, Pryse KM, Okunade AL, Su X. Fatty acid 2-hydroxylase mediates diffusional mobility of Raft-associated lipids, GLUT4 level, and lipogenesis in 3T3-L1 adipocytes. J Biol Chem. 2010. .CrossRefGoogle Scholar
  6. 6.
    Crown SB, Marze N, Antoniewicz MR. Catabolism of branched chain amino acids contributes significantly to synthesis of odd-chain and even-chain fatty acids in 3T3-L1 adipocytes. PLoS One. 2015. .CrossRefGoogle Scholar
  7. 7.
    Wang J, Wang C, Han X. Tutorial on lipidomics. Anal Chim Acta. 2019. .CrossRefGoogle Scholar
  8. 8.
    Li J, Vosegaard T, Guo Z. Applications of nuclear magnetic resonance in lipid analyses: an emerging powerful tool for lipidomics studies. Prog Lipid Res. 2017. .CrossRefGoogle Scholar
  9. 9.
    Meusel A, Popkova Y, Dannenberger D, Schiller J. A high-resolution NMR approach combined to MALDI-TOF-MS to estimate the positional distribution of acyl-linked unsaturated fatty acids in triacylglycerols. Food Anal Methods. 2017. .CrossRefGoogle Scholar
  10. 10.
    Gross JH. Mass Spectrometry: a textbook. 2nd ed. Berlin, Heidelberg: Springer-Verlag Berlin Heidelberg; 2011.CrossRefGoogle Scholar
  11. 11.
    Petkovic M, Schiller J, Müller M, Benard S, Reichl S, Arnold K, et al. Detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species. Anal Biochem. 2001. .CrossRefGoogle Scholar
  12. 12.
    Eibisch M, Fuchs B, Schiller J, Süß R, Teuber K. Analysis of phospholipid mixtures from biological tissues by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS): a laboratory experiment. J Chem Educ. 2011. .CrossRefGoogle Scholar
  13. 13.
    Hsu FF. Mass spectrometry-based shotgun lipidomics—a critical review from the technical point of view. Anal Bioanal Chem. 2018. .CrossRefGoogle Scholar
  14. 14.
    Jurowski K, Kochan K, Walczak J, Barańska M, Piekoszewski W, Buszewski B. Analytical techniques in lipidomics: state of the art. Crit Rev Anal Chem. 2017. .CrossRefGoogle Scholar
  15. 15.
    Fuchs B, Süss R, Teuber K, Eibisch M, Schiller J. Lipid analysis by thin-layer chromatography—a review of the current state. J Chromatogr A. 2011. .CrossRefGoogle Scholar
  16. 16.
    Griesinger H, Fuchs B, Süß R, Matheis K, Schulz M, Schiller J. Stationary phase thickness determines the quality of thin-layer chromatography/matrix-assisted laser desorption and ionization mass spectra of lipids. Anal Biochem. 2014. .CrossRefGoogle Scholar
  17. 17.
    Nimptsch A, Fuchs B, Süß R, Zschörnig K, Jakop U, Göritz F, et al. A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry. Anal Bioanal Chem. 2013. .CrossRefGoogle Scholar
  18. 18.
    Dodds ED, McCoy MR, Rea LD, Kennish JM. Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact mass spectrometry. Lipids. 2005. .CrossRefGoogle Scholar
  19. 19.
    Zebisch K, Voigt V, Wabitsch M, Brandsch M. Protocol for effective differentiation of 3T3-L1 cells to adipocytes. Anal Biochem. 2012. Scholar
  20. 20.
    Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res. 2008. CrossRefGoogle Scholar
  21. 21.
    Engel KM, Sampels S, Dzyuba B, Podhorec P, Policar T, Dannenberger D, et al. Swimming at different temperatures: The lipid composition of sperm from three freshwater fish species determined by mass spectrometry and nuclear magnetic resonance spectroscopy. Chem Phys Lipids. 2019. .CrossRefGoogle Scholar
  22. 22.
    Zhang S, Wang Y, Cui L, Deng Y, Xu S, Yu J, et al. Morphologically and functionally distinct lipid droplet subpopulations. Sci Rep. 2016. .
  23. 23.
    Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959. .CrossRefGoogle Scholar
  24. 24.
    Dannenberger D, Nuernberg G, Nuernberg K, Will K, Schauer N, Schmicke M. Effects of diets supplemented with n-3 or n-6 PUFA on pig muscle lipid metabolites measured by non-targeted LC-MS lipidomic profiling. J Food Compos Anal. 2017. CrossRefGoogle Scholar
  25. 25.
    Herdmann A, Martin J, Nuernberg G, Dannenberger D, Nuernberg K. Effect of dietary n-3 and n-6 PUFA on lipid composition of different tissues of German Holstein bulls and the fate of bioactive fatty acids during processing. J Agric Food Chem. 2010. CrossRefGoogle Scholar
  26. 26.
    Zhao X, Hu H, Wang C, Bai L, Wang Y, Wang W, et al. A comparison of methods for effective differentiation of the frozen-thawed 3T3-L1 cells. Anal Biochem. 2019. .
  27. 27.
    Fuchs B, Schiller J, Süss R, Schürenberg M, Suckau D. A direct and simple method of coupling matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis of phospholipids from egg yolk. Anal Bioanal Chem. 2007. .CrossRefGoogle Scholar
  28. 28.
    van Meer G, Voelker DR, Feigenson GW. Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol. 2008. .CrossRefGoogle Scholar
  29. 29.
    Renne MF, de Kroon AIPM. The role of phospholipid molecular species in determining the physical properties of yeast membranes. FEBS Lett. 2018. .CrossRefGoogle Scholar
  30. 30.
    Quehenberger O, Armando AM, Dennis EA. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry. Biochim Biophys Acta. 2011. .CrossRefGoogle Scholar
  31. 31.
    Arisawa K, Ichi I, Yasukawa Y, Sone Y, Fujiwara Y. Changes in the phospholipid fatty acid composition of the lipid droplet during the differentiation of 3T3-L1 adipocytes. J Biochem. 2013. .CrossRefGoogle Scholar
  32. 32.
    Ichi I, Kono N, Arita Y, Haga S, Arisawa K, Yamano M, et al. Identification of genes and pathways involved in the synthesis of Mead acid (20:3n-9), an indicator of essential fatty acid deficiency. Biochim Biophys Acta. 2014. .CrossRefGoogle Scholar
  33. 33.
    Mead JF, Slaton WH. Metabolism of essential fatty acids. III. Isolation of 5,8,11-eicosatrienoic acid from fat-deficient rats. J Biol Chem. 1956;219:705–9.PubMedGoogle Scholar
  34. 34.
    Li Y, Rong Y, Bao L, Nie B, Ren G, Zheng C, et al. Suppression of adipocyte differentiation and lipid accumulation by stearidonic acid (SDA) in 3T3-L1 cells. Lipids Health Dis. 2017. .
  35. 35.
    Kim YC, Gomez FE, Fox BG, Ntambi JM. Differential regulation of the stearoyl-CoA desaturase genes by thiazolidinediones in 3T3-L1 adipocytes. J Lipid Res. 2000;41:1310–6.PubMedGoogle Scholar
  36. 36.
    Ntambi JM, Miyazaki M, Stoehr JP, Lan H, Kendziorski CM, Yandell BS, et al. Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity. Proc Natl Acad Sci U S A. 2002. .CrossRefGoogle Scholar
  37. 37.
    Ralston JC, Mutch DM. SCD1 inhibition during 3T3-L1 adipocyte differentiation remodels triacylglycerol, diacylglycerol and phospholipid fatty acid composition. Prostaglandins Leukot Essent Fatty Acids. 2015. .CrossRefGoogle Scholar
  38. 38.
    Ralston JC, Badoud F, Cattrysse B, McNicholas PD, Mutch DM. Inhibition of stearoyl-CoA desaturase-1 in differentiating 3T3-L1 preadipocytes upregulates elongase 6 and downregulates genes affecting triacylglycerol synthesis. Int J Obes (Lond). 2014. .CrossRefGoogle Scholar
  39. 39.
    Green CD, Ozguden-Akkoc CG, Wang Y, Jump DB, Olson LK. Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species. J Lipid Res. 2010. .CrossRefGoogle Scholar
  40. 40.
    Burns TA, Kadegowda AKG, Duckett SK, Pratt SL, Jenkins TC. Palmitoleic (16:1 cis-9) and cis-vaccenic (18:1 cis-11) acid alter lipogenesis in bovine adipocyte cultures. Lipids. 2012. .CrossRefGoogle Scholar
  41. 41.
    Pilch PF, Thompson PA, Czech MP. Coordinate modulation of D-glucose transport activity and bilayer fluidity in plasma membranes derived from control and insulin-treated adipocytes. Proc Natl Acad Sci U S A. 1980. .CrossRefGoogle Scholar
  42. 42.
    Harayama T, Riezman H. Understanding the diversity of membrane lipid composition. Nat Rev Mol Cell Biol. 2018. .CrossRefGoogle Scholar
  43. 43.
    Halama A, Horsch M, Kastenmüller G, Möller G, Kumar P, Prehn C, et al. Metabolic switch during adipogenesis: from branched chain amino acid catabolism to lipid synthesis. Arch Biochem Biophys. 2016. .CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yulia Popkova
    • 1
    Email author
  • Dirk Dannenberger
    • 2
  • Jürgen Schiller
    • 1
  • Kathrin M. Engel
    • 1
  1. 1.Institute for Medical Physics and Biophysics, Medical FacultyLeipzig UniversityLeipzigGermany
  2. 2.Leibniz Institute for Farm Animal BiologyInstitute of Muscle Biology and GrowthDummerstorfGermany

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