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Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease

Abstract

Excess lipid accumulation in peripheral tissues is a key feature of many metabolic diseases. Therefore, techniques for imaging and quantifying lipids in various tissues are important for understanding and evaluating the overall metabolic status of a research subject. Here we present a protocol that detects neutral lipids and lipid droplet (LD) morphology by oil red O (ORO) staining of sections from frozen tissues. The method allows for easy estimation of tissue lipid content and distribution using only basic laboratory and computer equipment. Furthermore, the procedure described here is well suited for the comparison of different metabolically challenged animal models. As an example, we include data on muscular and hepatic lipid accumulation in diet-induced and genetically induced diabetic mice. The experimental description presents details for optimal staining of lipids using ORO, including tissue collection, sectioning, staining, imaging and measurements of tissue lipids, in a time frame of less than 2 d.

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Figure 1: Neutral lipid accumulation in tissues from metabolically challenged mice.

References

  1. Samuel, V.T., Petersen, K.F. & Shulman, G.I. Lipid-induced insulin resistance: unravelling the mechanism. Lancet 375, 2267–2277 (2010).

    Article  CAS  Google Scholar 

  2. Hagberg, C.E. et al. Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes. Nature 490, 426–430 (2012).

    Article  CAS  Google Scholar 

  3. Unger, R.H. Lipotoxic diseases. Annu. Rev. Med. 53, 319–336 (2002).

    Article  CAS  Google Scholar 

  4. Yu, C. et al. 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 (2002).

    Article  CAS  Google Scholar 

  5. Bostrom, P. et al. SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity. Nat. Cell Biol 9, 1286–1293 (2007).

    Article  Google Scholar 

  6. Sollner, T.H. Lipid droplets highjack SNAREs. Nat. Cell Biol. 9, 1219–1220 (2007).

    Article  Google Scholar 

  7. Kim, F. et al. Vascular inflammation, insulin resistance, and reduced nitric oxide production precede the onset of peripheral insulin resistance. Arterioscler. Thromb. Vasc. Biol. 28, 1982–1988 (2008).

    Article  CAS  Google Scholar 

  8. Duncan, E.R. et al. Effect of endothelium-specific insulin resistance on endothelial function in vivo. Diabetes 57, 3307–3314 (2008).

    Article  CAS  Google Scholar 

  9. Park, S.Y. et al. Unraveling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice. Diabetes 54, 3530–3540 (2005).

    Article  CAS  Google Scholar 

  10. Ahmadian, M., Duncan, R.E. & Sul, H.S. The skinny on fat: lipolysis and fatty acid utilization in adipocytes. Trends Endocrinol. Metab. 20, 424–428 (2009).

    Article  CAS  Google Scholar 

  11. Farese, R.V. Jr., Zechner, R., Newgard, C.B. & Walther, T.C. The problem of establishing relationships between hepatic steatosis and hepatic insulin resistance. Cell Metab. 15, 570–573 (2012).

    Article  CAS  Google Scholar 

  12. Avramoglu, R.K., Basciano, H. & Adeli, K. Lipid and lipoprotein dysregulation in insulin resistant states. Clin. Chim. Acta. 368, 1–19 (2006).

    Article  CAS  Google Scholar 

  13. Hagberg, C.E. et al. Vascular endothelial growth factor B controls endothelial fatty acid uptake. Nature 464, 917–921 (2010).

    Article  CAS  Google Scholar 

  14. Spangenburg, E.E., Pratt, S.J., Wohlers, L.M. & Lovering, R.M. Use of BODIPY (493/503) to visualize intramuscular lipid droplets in skeletal muscle. J. Biomed. Biotechnol. 2011, 598358 (2011).

    Article  Google Scholar 

  15. Falcon, A. et al. FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase. Am. J. Physiol. Endocrinol. Metab. 299, E384–E393 (2010).

    Article  CAS  Google Scholar 

  16. Bonilla, E. & Prelle, A. Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle. J. Histochem. Cytochem. 35, 619–621 (1987).

    Article  CAS  Google Scholar 

  17. Fowler, S.D. & Greenspan, P. Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O. J. Histochem. Cytochem. 33, 833–836 (1985).

    Article  CAS  Google Scholar 

  18. Fuchs, B., Suss, R., Teuber, K., Eibisch, M. & Schiller, J. Lipid analysis by thin-layer chromatography—a review of the current state. J. Chromatogr. A 1218, 2754–2774 (2011).

    Article  CAS  Google Scholar 

  19. Fuchs, B., Suss, R. & Schiller, J. An update of MALDI-TOF mass spectrometry in lipid research. Prog. Lipid Res. 49, 450–475 (2010).

    Article  CAS  Google Scholar 

  20. Ramirez-Zacarias, J.L., Castro-Munozledo, F. & Kuri-Harcuch, W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 97, 493–497 (1992).

    Article  CAS  Google Scholar 

  21. Levene, A.P. et al. Quantifying hepatic steatosis—more than meets the eye. Histopathology 60, 971–981 (2012).

    Article  Google Scholar 

  22. Catta-Preta, M., Mendonca, L.S., Fraulob-Aquino, J., Aguila, M.B. & Mandarim-de-Lacerda, C.A. A critical analysis of three quantitative methods of assessment of hepatic steatosis in liver biopsies. Virchows Arch. 459, 477–485 (2011).

    Article  Google Scholar 

  23. De Bock, K. et al. Evaluation of intramyocellular lipid breakdown during exercise by biochemical assay, NMR spectroscopy, and Oil Red O staining. Am. J. Physiol. Endocrinol. Metab. 293, E428–E434 (2007).

    Article  CAS  Google Scholar 

  24. Shaw, C.S., Jones, D.A. & Wagenmakers, A.J. Network distribution of mitochondria and lipid droplets in human muscle fibres. Histochem. Cell Biol. 129, 65–72 (2008).

    Article  CAS  Google Scholar 

  25. Olofsson, S.O. et al. Triglyceride containing lipid droplets and lipid droplet-associated proteins. Curr. Opin. Lipidol. 19, 441–447 (2008).

    Article  CAS  Google Scholar 

  26. Fukumoto, S. & Fujimoto, T. Deformation of lipid droplets in fixed samples. Histochem. Cell Biol. 118, 423–428 (2002).

    Article  CAS  Google Scholar 

  27. Zhou, J., Lhotak, S., Hilditch, B.A. & Austin, R.C. Activation of the unfolded protein response occurs at all stages of atherosclerotic lesion development in apolipoprotein E–deficient mice. Circulation 111, 1814–1821 (2005).

    Article  CAS  Google Scholar 

  28. Pizzurro, G.A. et al. High lipid content of irradiated human melanoma cells does not affect cytokine-matured dendritic cell function. Cancer Immunol. Immunother. 62, 3–15 (2013).

    Article  CAS  Google Scholar 

  29. Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  Google Scholar 

  30. Geerts, A. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin. Liver Dis. 21, 311–335 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We want to thank S. Wittgren, E. Cortez and C. Mössinger for technical assistance. This study was supported by The Novo Nordisk Foundation, The Swedish Cancer Foundation, The Swedish Research Council, Torsten och Ragnar Söderbergs Stiftelser, The Peter Wallenberg Foundation for Economics and Technology, The Swedish Heart and Lung Foundation, Ludwig Institute for Cancer Research and Karolinska Institutet. C.E.H. was kindly supported by Frans Wilhelm och Waldemar von Frenckells Fond and by Wilhelm och Else Stockmanns Stiftelse.

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Authors

Contributions

A.M. performed in vivo mouse experiments, ORO analyses and wrote the paper; C.E.H. optimized the protocol and commented on the paper; L.M. commented on the paper; U.E. commented on the paper; A.F. optimized the protocol, performed ORO analyses, assisted in in vivo mouse studies and wrote the paper. All the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Annelie Falkevall.

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The authors declare no competing financial interests.

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Mehlem, A., Hagberg, C., Muhl, L. et al. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc 8, 1149–1154 (2013). https://doi.org/10.1038/nprot.2013.055

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