Skip to main content
SpringerLink
Log in

Measurements of Diacylglycerols in Skeletal Muscle by Atmospheric Pressure Chemical Ionization Mass Spectrometry

  • Methods
  • Published:
Lipids

Abstract

Diacylglycerol (DAG) is an intermediate lipid involved in the synthesis of phospholipids and triglycerides. As signaling regulators, DAG activate novel protein kinase C leading to decreased response to insulin in skeletal muscle. Alteration of DAG contents correlates with development of metabolic dysregulation in obese and diabetic conditions. Recent advances in lipidomics using mass spectrometry allow expanded measurements of various lipid species. This study describes a rapid measurement of DAG species using the triple quadrupole mass spectrometry using atmospheric pressure chemical ionization in a positive ion mode. DAG in the cells and muscle tissues were separated depending on differences in chain lengths and degree of unsaturation. The limit of detection and quantification for DAG was 0.2 to 17 pmol for this method. When C2C12 cells were treated with palmitate or oleate, we found a 12-fold and 2-fold DAG increase respectively compared to the no-treatment control. In the muscles of obese db/db mice, DAG levels were elevated by 6-fold compared to those of wild-type skeletal muscles. The present analytical method provides a rapid and sensitive quantification of DAG molecular species from various biological samples and can be used to correlate the degree of metabolic dysregulation with lipotoxic metabolites.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

APCI:

Atmospheric pressure chemical ionization

DAG:

Diacylglycerol(s)

ELSD:

Evaporative light scattering detector

FFA:

Free fatty acid(s)

HPLC:

High performance liquid chromatography

MS/MS:

Tandem mass spectrometry

PBS:

Phosphate-buffered saline

PKC:

Protein kinase C

References

  1. Zimmet P, Alberti KG, Shaw J (2001) Global and societal implications of the diabetes epidemic. Nature 414:782–787

    Article  PubMed  CAS  Google Scholar 

  2. Young LR, Nestle M (2002) The contribution of expanding portion sizes to the US obesity epidemic. Am J Public Health 92:246–249

    Article  PubMed  Google Scholar 

  3. Shulman GI (2000) Cellular mechanisms of insulin resistance. J Clin Invest 106:171–176

    Article  PubMed  CAS  Google Scholar 

  4. Laakso M, Voutilainen E, Sarlund H, Aro A, Pyorala K, Penttila I (1985) Serum lipids and lipoproteins in middle-aged non-insulin-dependent diabetics. Atherosclerosis 56:271–281

    Article  PubMed  CAS  Google Scholar 

  5. Boden G (2003) Effects of free fatty acids (FFA) on glucose metabolism: significance for insulin resistance and type 2 diabetes. Exp Clin Endocrinol Diabetes 111:121–124

    Article  PubMed  CAS  Google Scholar 

  6. Lewis B, Mancini M, Mattock M, Chait A, Fraser TR (1972) Plasma triglyceride and fatty acid metabolism in diabetes mellitus. Eur J Clin Invest 2:445–453

    Article  PubMed  CAS  Google Scholar 

  7. Holland WL, Summers SA (2008) Sphingolipids, insulin resistance, and metabolic disease: new insights from in vivo manipulation of sphingolipid metabolism. Endocr Rev 29:381–402

    Article  PubMed  CAS  Google Scholar 

  8. Samuel VT, Petersen KF, Shulman GI (2010) Lipid-induced insulin resistance: unravelling the mechanism. Lancet 375:2267–2277

    Article  PubMed  CAS  Google Scholar 

  9. Perry DK, Bielawska A, Hannun YA (2000) Quantitative determination of ceramide using diglyceride kinase. Methods Enzymol 312:22–31

    Article  PubMed  CAS  Google Scholar 

  10. Pacheco YM, Perez-Camino MC, Cert A, Montero E, Ruiz-Gutierrez V (1998) Determination of the molecular species composition of diacylglycerols in human adipose tissue by solid-phase extraction and gas chromatography on a polar phase. J Chromatogr B Biomed Sci Appl 714:127–132

    Article  PubMed  CAS  Google Scholar 

  11. Graeve M, Janssen D (2009) Improved separation and quantification of neutral and polar lipid classes by HPLC-ELSD using a monolithic silica phase: application to exceptional marine lipids. J Chromatogr B Analyt Technol Biomed Life Sci 877:1815–1819

    Article  PubMed  CAS  Google Scholar 

  12. Lee C, Fisher SK, Agranoff BW, Hajra AK (1991) Quantitative analysis of molecular species of diacylglycerol and phosphatidate formed upon muscarinic receptor activation of human SK-N-SH neuroblastoma cells. J Biol Chem 266:22837–22846

    PubMed  CAS  Google Scholar 

  13. Striby L, Lafont R, Goutx M (1999) Improvement in the Iatroscan thin-layer chromatographic-flame ionisation detection analysis of marine lipids. Separation and quantitation of monoacylglycerols and diacylglycerols in standards and natural samples. J Chromatogr A 849:371–380

    Article  PubMed  CAS  Google Scholar 

  14. Leiker TJ, Barkley RM, Murphy RC (2011) Analysis of diacylglycerol molecular species in cellular lipid extracts by normal-phase LC-electrospray mass spectrometry. Int J Mass Spectrom 305:103–109

    Article  PubMed  CAS  Google Scholar 

  15. Callender HL, Forrester JS, Ivanova P, Preininger A, Milne S, Brown HA (2007) Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm. Anal Chem 79:263–272

    Article  PubMed  CAS  Google Scholar 

  16. Li YL, Su X, Stahl PD, Gross ML (2007) Quantification of diacylglycerol molecular species in biological samples by electrospray ionization mass spectrometry after one-step derivatization. Anal Chem 79:1569–1574

    Article  PubMed  CAS  Google Scholar 

  17. Murphy RC, James PF, McAnoy AM, Krank J, Duchoslav E, Barkley RM (2007) Detection of the abundance of diacylglycerol and triacylglycerol molecular species in cells using neutral loss mass spectrometry. Anal Biochem 366:59–70

    Article  PubMed  CAS  Google Scholar 

  18. Ota T, Gayet C, Ginsberg HN (2008) Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents. J Clin Invest 118:316–332

    Article  PubMed  CAS  Google Scholar 

  19. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509

    PubMed  CAS  Google Scholar 

  20. Wendel AA, Lewin TM, Coleman RA (2009) Glycerol-3-phosphate acyltransferases: rate limiting enzymes of triacylglycerol biosynthesis. Biochim Biophys Acta 1791:501–506

    Article  PubMed  CAS  Google Scholar 

  21. Hsu FF, Ma Z, Wohltmann M, Bohrer A, Nowatzke W, Ramanadham S, Turk J (2000) Electrospray ionization/mass spectrometric analyses of human promonocytic U937 cell glycerolipids and evidence that differentiation is associated with membrane lipid composition changes that facilitate phospholipase A2 activation. J Biol Chem 275:16579–16589

    Article  PubMed  CAS  Google Scholar 

  22. Hsu FF, Bohrer A, Wohltmann M, Ramanadham S, Ma Z, Yarasheski K, Turk J (2000) Electrospray ionization mass spectrometric analyses of changes in tissue phospholipid molecular species during the evolution of hyperlipidemia and hyperglycemia in Zucker diabetic fatty rats. Lipids 35:839–854

    Article  PubMed  CAS  Google Scholar 

  23. Thyfault JP, Cree MG, Tapscott EB, Bell JA, Koves TR, Ilkayeva O, Wolfe RR, Dohm GL, Muoio DM (2010) Metabolic profiling of muscle contraction in lean compared with obese rodents. Am J Physiol Regul Integr Comp Physiol 299:R926–R934

    Article  PubMed  CAS  Google Scholar 

  24. Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, Bergeron R, Kim JK, Cushman SW, Cooney GJ, Atcheson B, White MF, Kraegen EW, Shulman GI (2002) Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 277:50230–50236

    Article  PubMed  CAS  Google Scholar 

  25. Niizeki T, Takeishi Y, Kitahara T, Arimoto T, Koyama Y, Goto K, Mende U, Kubota I (2008) Diacylglycerol kinase zeta rescues G alpha q-induced heart failure in transgenic mice. Circ J 72:309–317

    Article  PubMed  CAS  Google Scholar 

  26. Ryu D, Oh KJ, Jo HY, Hedrick S, Kim YN, Hwang YJ, Park TS, Han JS, Choi CS, Montminy M, Koo SH (2009) TORC2 regulates hepatic insulin signaling via a mammalian phosphatidic acid phosphatase, LIPIN1. Cell Metab 9:240–251

    Article  PubMed  CAS  Google Scholar 

  27. Savage DB, Petersen KF, Shulman GI (2007) Disordered lipid metabolism and the pathogenesis of insulin resistance. Physiol Rev 87:507–520

    Article  PubMed  CAS  Google Scholar 

  28. Liu L, Zhang Y, Chen N, Shi X, Tsang B, Yu YH (2007) Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance. J Clin Invest 117:1679–1689

    Article  PubMed  CAS  Google Scholar 

  29. Turban S, Hajduch E (2010) Protein kinase C isoforms: mediators of reactive lipid metabolites in the development of insulin resistance. FEBS Lett 585:269–274

    Article  PubMed  Google Scholar 

  30. Goni FM, Alonso A (1999) Structure and functional properties of diacylglycerols in membranes. Prog Lipid Res 38:1–48

    Article  PubMed  CAS  Google Scholar 

  31. Khan RS, Drosatos K, Goldberg IJ (2010) Creating and curing fatty hearts. Curr Opin Clin Nutr Metab Care 13:145–149

    Article  PubMed  Google Scholar 

  32. Idris I, Gray S, Donnelly R (2001) Protein kinase C activation: isozyme-specific effects on metabolism and cardiovascular complications in diabetes. Diabetologia 44:659–673

    Article  PubMed  CAS  Google Scholar 

  33. Werner ED, Lee J, Hansen L, Yuan M, Shoelson SE (2004) Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302. J Biol Chem 279:35298–35305

    Article  PubMed  CAS  Google Scholar 

  34. Powell DJ, Turban S, Gray A, Hajduch E, Hundal HS (2004) Intracellular ceramide synthesis and protein kinase Czeta activation play an essential role in palmitate-induced insulin resistance in rat L6 skeletal muscle cells. Biochem J 382:619–629

    Article  PubMed  CAS  Google Scholar 

  35. Watson ML, Coghlan M, Hundal HS (2009) Modulating serine palmitoyl transferase (SPT) expression and activity unveils a crucial role in lipid-induced insulin resistance in rat skeletal muscle cells. Biochem J 417:791–801

    Article  PubMed  CAS  Google Scholar 

  36. Holcapek M, Jandera P, Zderadicka P, Hruba L (2003) Characterization of triacylglycerol and diacylglycerol composition of plant oils using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. J Chromatogr A 1010:195–215

    Article  PubMed  CAS  Google Scholar 

  37. Montell E, Turini M, Marotta M, Roberts M, Noe V, Ciudad CJ, Mace K, Gomez-Foix AM (2001) DAG accumulation from saturated fatty acids desensitizes insulin stimulation of glucose uptake in muscle cells. Am J Physiol Endocrinol Metab 280:E229–E237

    PubMed  CAS  Google Scholar 

  38. Chavez JA, Summers SA (2003) Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys 419:101–109

    Article  PubMed  CAS  Google Scholar 

  39. Ryu D, Seo WY, Yoon YS, Kim YN, Kim SS, Kim HJ, Park TS, Choi CS, Koo SH (2011) Endoplasmic reticulum stress promotes LIPIN2-dependent hepatic insulin resistance. Diabetes 60:1072–1081

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF2010-0022120).

Conflict of interest

The authors declare no conflict of interests.

Author information

Authors and Affiliations

  1. Department of Life Science, Gachon University, Jinrikwan 304B, Bokjung-dong, Sujung-gu, Sungnam-Si, Gyeonggi-do, 461-701, Republic of Korea

    Su-Yeon Lee, Jung Ran Kim & Tae-Sik Park

  2. Nonghyup Food Safety Research Institute, Seoul, 137-130, Republic of Korea

    Mi-Young Ha

  3. Department of Food Science and Technology and Carbohydrate Bioproduct Research Center, Sejong University, Seoul, 143-747, Republic of Korea

    Soon-Mi Shim

Authors
  1. Su-Yeon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jung Ran Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mi-Young Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Soon-Mi Shim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tae-Sik Park
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tae-Sik Park.

Additional information

S.-Y. Lee and J. R. Kim contributed equally to this work.

About this article

Cite this article

Lee, SY., Kim, J.R., Ha, MY. et al. Measurements of Diacylglycerols in Skeletal Muscle by Atmospheric Pressure Chemical Ionization Mass Spectrometry. Lipids 48, 287–296 (2013). https://doi.org/10.1007/s11745-013-3766-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-013-3766-6

Keywords

Search

Navigation