Research

Biochemistry and Cell Biology of Lipids

Lipids are central for life and their importance as active players in Cell Biology and Physiology has sparked a high scientific interest in the last years. Lipid involvement in many cellular and organismal aspects has therefore triggered research in various fields.

The fact that the cellular lipid pool consists of several hundred individual lipid species is expression for the multitude of lipid functions and the challenge to understand their individual roles.

Lipid metabolic tracing – studying the dynamics of the lipidome

Why?

Fatty acids are major building blocks of membrane lipids and precursors of many signaling substances. In the form of triglycerides, they are abundant components of our nutrition. Major organs of fatty acid metabolism are gut, liver, muscle and adipose tissue, but all other tissues also have the capacity to use fatty acids either for generation of membrane lipids or of metabolic energy. Our lipidome contains several thousands of lipids, most of which contain fatty acids. Accordingly, the metabolism of fatty acids is extremely complex, and it is amazingly fast – a labeled fatty acid is found in hundreds of different compounds after five minutes of metabolism. Understanding this complexity and its pathological deviations needs experimental tools that offer high sensitivity and time resolution.

How?

Tracing needs a tracer that is as similar as possible to the target molecule but nonetheless reliably distinguishable. Our tracers are alkynes -  fatty acids and other lipids that contain a terminal triple bond. We feed them to biological systems in which they are metabolized, and then we collect and analyze the alkyne-containing (= labeled) lipids. For that, we have developed two technologies:

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Mass spectrometric tracing

This is the new approach published in 2019 (Thiele et al. 2019). Extracted alkyne lipids are reacted with the reporter C171.

C171 is optimized for improved detection by mass spectrometry. The positive charge strongly improves ionization, and the trialkylammonium group shows predictable fragmentation in tandem MS2.

Key advantages are:

  • Perfect specificity: labeled species are reliably discriminated from unlabeled species even if present in trace amounts
  • Strongly increased sensitivity: improved ionization results in improved sensitivity, typically by 5 – 50-fold.
  • Absolut quantification by addition of internal standards

Typical performance (75000 hepatocytes per sample):

  • Upon a 5 min labeling, identification of 150 – 250 labeled species
  • Upon a 1 h labeling, identification of up to 1000 labeled species
  • 15 – 20 lipid classes, 10 with absolute quantification

Single cell analysis:

  • Hepatocytes labeled for 3 h
  • Single cells by limiting dilution
  • 60 – 100 labeled species with absolute quantification

Multiplexing:

A set of deuterated versions of the reagent, the C175 reagents, enables a multiplexed version of the procedure

Additional advantages of multiplexing:

  • Four fold increased sample turnover
  • Improved sample-to-sample comparison by elimination of stochastic variation

Further reading:

Multiplexed and single cell tracing of lipid metabolism: A step by step protocol. Nature Protocol Exchange

And the Behind the Paper blog at Nature’s Protocol and Methods Community

Fluorescent tracing

By reaction with a fluorogenic coumarin dye, labeled lipids become fluorescent. After separation by TLC, the labeled spots can be identified by co-migrating external standards and quantified relative to each other. Absolute quantification is possible but not as reliable as in mass spectrometry.

Key advantages:

  • Easily accessible
  • Parallel sample processing with high throughput

This method was originally published in 2012 (Thiele et al. 2012) and has since then been used in numerous publications:

Publication bibliography

Salo, Veijo T.; Li, Shiqian; Vihinen, Helena; Hölttä-Vuori, Maarit; Szkalisity, Abel; Horvath, Peter et al. (2019): Seipin Facilitates Triglyceride Flow to Lipid Droplet and Counteracts Droplet Ripening via Endoplasmic Reticulum Contact. In Dev. Cell 50 (4), 478-493.e9. DOI: 10.1016/j.devcel.2019.05.016.

Luukkonen, Panu K.; Nick, Auli; Hölttä-Vuori, Maarit; Thiele, Christoph; Isokuortti, Elina; Lallukka-Brück, Susanna et al. (2019): Human PNPLA3-I148M variant increases hepatic retention of polyunsaturated fatty acids. In JCI insight 4 (16). DOI: 10.1172/jci.insight.127902.

Segerer, Gabriela; Engelmann, Daria; Kaestner, Alexandra; Trötzmüller, Martin; Köfeler, Harald; Stigloher, Christian et al. (2018): A phosphoglycolate phosphatase/AUM-dependent link between triacylglycerol turnover and epidermal growth factor signaling. In Biochimica et biophysica acta. Molecular and cell biology of lipids 1863 (6), pp. 584–594. DOI: 10.1016/j.bbalip.2018.03.002.

Hofmann, Kristina; Lamberz, Christian; Piotrowitz, Kira; Offermann, Nina; But, Diana; Scheller, Anja et al. (2017): Tanycytes and a differential fatty acid metabolism in the hypothalamus. In Glia 65 (2), pp. 231–249. DOI: 10.1002/glia.23088.

Alecu, Irina; Tedeschi, Andrea; Behler, Natascha; Wunderling, Klaus; Lamberz, Christian; Lauterbach, Mario A. R. et al. (2017): Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction. In Journal of lipid research 58 (1), pp. 42–59. DOI: 10.1194/jlr.M068676.

Salo, Veijo T.; Belevich, Ilya; Li, Shiqian; Karhinen, Leena; Vihinen, Helena; Vigouroux, Corinne et al. (2016): Seipin regulates ER-lipid droplet contacts and cargo delivery. In The EMBO journal 35 (24), pp. 2699–2716. DOI: 10.15252/embj.201695170.

Merklinger, Elisa; Schloetel, Jan-Gero; Spitta, Luis; Thiele, Christoph; Lang, Thorsten (2016): No Evidence for Spontaneous Lipid Transfer at ER-PM Membrane Contact Sites. In The Journal of membrane biology 249 (1-2), pp. 41–56. DOI: 10.1007/s00232-015-9845-2.

Gaebler, Anne; Penno, Anke; Kuerschner, Lars; Thiele, Christoph (2016): A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins. In Journal of lipid research 57 (10), pp. 1934–1947. DOI: 10.1194/jlr.D070565.

Wolf, Monika Julia; Adili, Arlind; Piotrowitz, Kira; Abdullah, Zeinab; Boege, Yannick; Stemmer, Kerstin et al. (2014): Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes. In Cancer cell 26 (4), pp. 549–564. DOI: 10.1016/j.ccell.2014.09.003.

Schneider, Christoph; Nobs, Samuel P.; Kurrer, Michael; Rehrauer, Hubert; Thiele, Christoph; Kopf, Manfred (2014): Induction of the nuclear receptor PPAR-γ by the cytokine GM-CSF is critical for the differentiation of fetal monocytes into alveolar macrophages. In Nature immunology 15 (11), pp. 1026–1037. DOI: 10.1038/ni.3005.

Moessinger, Christine; Klizaite, Kristina; Steinhagen, Almut; Philippou-Massier, Julia; Shevchenko, Andrej; Hoch, Michael et al. (2014): Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage. In BMC cell biology 15, p. 43. DOI: 10.1186/s12860-014-0043-3.

Itoe, Maurice A.; Sampaio, Júlio L.; Cabal, Ghislain G.; Real, Eliana; Zuzarte-Luis, Vanessa; March, Sandra et al. (2014): Host cell phosphatidylcholine is a key mediator of malaria parasite survival during liver stage infection. In Cell host & microbe 16 (6), pp. 778–786. DOI: 10.1016/j.chom.2014.11.006.

Hofmann, Kristina; Thiele, Christoph; Schött, Hans-Frieder; Gaebler, Anne; Schoene, Mario; Kiver, Yuriy et al. (2014): A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization. In Journal of lipid research 55 (3), pp. 583–591. DOI: 10.1194/jlr.D044727.

Gaebler, Anne; Milan, Robin; Straub, Leon; Hoelper, Dominik; Kuerschner, Lars; Thiele, Christoph (2013): Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays. In Journal of lipid research 54 (8), pp. 2282–2290. DOI: 10.1194/jlr.D038653.

Kuerschner, Lars; Richter, Doris; Hannibal-Bach, Hans Kristian; Gaebler, Anne; Shevchenko, Andrej; Ejsing, Christer S.; Thiele, Christoph (2012): Exogenous ether lipids predominantly target mitochondria. In PloS one 7 (2), e31342. DOI: 10.1371/journal.pone.0031342.

Thiele, Christoph; Papan, Cyrus; Hoelper, Dominik; Kusserow, Kalina; Gaebler, Anne; Schoene, Mario et al. (2012): Tracing fatty acid metabolism by click chemistry. In ACS chemical biology 7 (12), pp. 2004–2011. DOI: 10.1021/cb300414v.

Immunoregulation by lipids

  • Liver lipid metabolism

Nonalcoholic steatohepatitis (NASH) is a severe liver condition also leading to hepatocellular carcinoma (HCC), the fastest rising cancer in the United States and also increasing in Europe. The mechanisms underlying NASH and NASH-induced HCC are largely unknown. 
   A mouse model featuring long-term feeding of a choline-deficient high-fat diet successfully recapitulates key features of human metabolic syndrome, NASH, and HCC. As in patients, induction of activated intrahepatic CD8+ T cells, NKT cells, and inflammatory cytokines are a hallmark. We could demonstrate that the NKT cells profoundly influence the lipid metabolism in hepatocytes. These intrahepatic cell interactions involve LTßR and canonical NF-κB signaling and facilitate NASH-to-HCC transition (Wolf et al. 2014).

Lipid droplets - Function, regulation and composition

  • Local lipid metabolism and protein degradation

Lipid droplets (LDs) are the main cellular site for neutral lipid storage but are also important for lipid metabolism, proteasomal protein degradation and autophagy. 
   We have extensively studied the LD organelle and its lipid and protein content (Spandl et al. 2008)(Grimard et al. 2008). We were the first to demonstrate the presence of the enzymes diacylglycerol-acyltransferase 2 (DGAT2) and lyso-phosphatidyl-acyltransferase1/2 (LPCAT1/2) at LDs and to show that these enzymes locally synthesize neutral and phospholipids, respectively (Kuerschner et al. 2008)(Moessinger et al. 2011)
   It was concluded that LDs have the capacity to adapt their surface to volume ratio by local synthesis of neutral and phospholipids independent of direct connections to the endoplasmic reticulum (Penno et al. 2013)(Moessinger et al. 2014)
   We also found the ancient ubiquitous protein 1 (AUP1), a protein involved in proteasomal protein degradation, localizing to LDs in a monotopic fashionhttp://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072453. Locally, the highly conserved AUP1 engages in a complex with the Ube2g2 protein and thereby establishes a direct molecular link between the LD organelle and the cellular ubiquitination machinery (Spandl et al. 2011)(Stevanovic et al. 2013)(Lohmann et al 2013).

Lipid-protein interactions

  • Photoactivatable lipid probes

Although all membrane proteins closely interact with lipids, these protein-lipid interactions are only infrequently studied due to technical challenges. Our photoactivatable lipids yielded important new insight into the role of lipids in membrane traffic (Suchanek et al. 2007Thiele et al. 2000Schmidt et al. 1999)
   Using our 'photocholesterol' we were able to prove the interaction of oxysterol-binding protein related proteins (OPRs) and cholesterol. A detailed picture of the active cholesterol-binding residues was drawn by the combination of cholesterol-photocrosslinking assays and site-directed mutagenesis of ORP2 (Suchanek et al. 2007)
   The 'polyene lipids' developed in our lab (Kuerschner et al. 2005) have also been used as fluorescent lipid probes to assess lipid-lipid and lipid-protein interactions by FRET analysis (Ernst et al. 2012)(Contreras et al. 2012)

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