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European Journal of Nutrition

, Volume 58, Issue 8, pp 3023–3034 | Cite as

Differential capability of metabolic substrates to promote hepatocellular lipid accumulation

  • Ngoc Anh Hoang
  • Friederike Richter
  • Martin Schubert
  • Stefan Lorkowski
  • Lars-Oliver Klotz
  • Holger SteinbrennerEmail author
Original Contribution

Abstract

Purpose

Excessive storage of triacylglycerides (TAGs) in lipid droplets within hepatocytes is a hallmark of non-alcoholic fatty liver disease (NAFLD), one of the most widespread metabolic disorders in Western societies. For the purpose of exploring molecular pathways in NAFLD development and testing potential drug candidates, well-characterised experimental models of ectopic TAG storage in hepatocytes are needed.

Methods

Using an optimised Oil Red O assay, immunoblotting and real-time qRT-PCR, we compared the capability of dietary monosaccharides and fatty acids to promote lipid accumulation in HepG2 human hepatoma cells.

Results

Both high glucose and high fructose resulted in intracellular lipid accumulation after 48 h, and this was further augmented (up to twofold, as compared to basal levels) by co-treatment with the lipogenesis-stimulating hormone insulin and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α), respectively. The fatty acids palmitic and oleic acid were even more effective than these carbohydrates, inducing significantly elevated TAG storage already after 24 h of treatment. Highest (about threefold) increases in lipid accumulation were observed upon treatment with oleic acid, alone as well as in combinations with palmitic acid or with high glucose and insulin. Increases in protein levels of a major lipid droplet coat protein, perilipin-2 (PLIN2), mirrored intracellular lipid accumulation following different treatment regimens.

Conclusions

Several treatment regimens of excessive fat and sugar supply promoted lipid accumulation in HepG2 cells, albeit with differences in the extent and rapidity of steatogenesis. PLIN2 is a candidate molecular marker of sustained lipid accumulation in HepG2 cells.

Keywords

Fatty liver Triglyceride Glucosamine Adipophilin DGAT2 

Abbreviations

ACC

Acetyl-CoA carboxylase

BSA

Bovine serum albumin

CV

Coefficient of variation

ChREBP

Carbohydrate-responsive element-binding protein

DGAT

Diacylglycerol O-acyltransferase

DMEM

Dulbecco’s modified Eagle’s medium

DNL

De novo lipogenesis

ER

Endoplasmic reticulum

FAS

Fatty acid synthase

FBS

Foetal bovine serum

Frc

Fructose

GFAT

Glutamine–fructose-6-phosphate aminotransferase

Glc

Glucose

GlcN

Glucosamine

HBP

Hexosamine biosynthesis pathway

Ins

Insulin

Man

Mannitol

MTT

Thiazolyl blue tetrazolium bromide

NAFLD

Non-alcoholic fatty liver disease

NHANES

National Health and Nutrition Examination Survey

NEFAs

Non-esterified fatty acids

OA

Oleic acid

ORO

Oil Red O

PA

Palmitic acid

PLIN2

Perilipin-2 (adipophilin)

PPAR-γ

Peroxisome proliferator-activated receptor gamma

SREBP-1

Sterol regulatory element-binding protein 1

TAG

Triacylglyceride

TNF-α

Tumour necrosis factor alpha

VLDL

Very low density lipoproteins

Notes

Acknowledgements

We thank K. Erler for excellent technical assistance and F. Begett for her help with the RT-qPCR analyses.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

394_2018_1847_MOESM1_ESM.pdf (358 kb)
Supplementary material 1 (PDF 357 KB)

References

  1. 1.
    Bellentani S, Scaglioni F, Marino M, Bedogni G (2010) Epidemiology of non-alcoholic fatty liver disease. Dig Dis 28(1):155–161.  https://doi.org/10.1159/000282080 CrossRefPubMedGoogle Scholar
  2. 2.
    Reccia I, Kumar J, Akladios C, Virdis F, Pai M, Habib N, Spalding D (2017) Non-alcoholic fatty liver disease: a sign of systemic disease. Metabolism 72:94–108.  https://doi.org/10.1016/j.metabol.2017.04.011 CrossRefPubMedGoogle Scholar
  3. 3.
    Kawano Y, Cohen DE (2013) Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver disease. J Gastroenterol 48(4):434–441.  https://doi.org/10.1007/s00535-013-0758-5 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Le MH, Devaki P, Ha NB, Jun DW, Te HS, Cheung RC, Nguyen MH (2017) Prevalence of non-alcoholic fatty liver disease and risk factors for advanced fibrosis and mortality in the United States. PLoS One 12(3):e0173499.  https://doi.org/10.1371/journal.pone.0173499 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ (2005) Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Investig 115(5):1343–1351.  https://doi.org/10.1172/JCI23621 CrossRefPubMedGoogle Scholar
  6. 6.
    Hsu CC, Ness E, Kowdley KV (2017) Nutritional approaches to achieve weight loss in nonalcoholic fatty liver disease. Adv Nutr 8(2):253–265.  https://doi.org/10.3945/an.116.013730 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Softic S, Cohen DE, Kahn CR (2016) Role of dietary fructose and hepatic de novo lipogenesis in fatty liver disease. Dig Dis Sci 61(5):1282–1293.  https://doi.org/10.1007/s10620-016-4054-0 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Spruss A, Bergheim I (2009) Dietary fructose and intestinal barrier: potential risk factor in the pathogenesis of nonalcoholic fatty liver disease. J Nutr Biochem 20(9):657–662.  https://doi.org/10.1016/j.jnutbio.2009.05.006 CrossRefPubMedGoogle Scholar
  9. 9.
    Kanuri G, Bergheim I (2013) In vitro and in vivo models of non-alcoholic fatty liver disease (NAFLD). Int J Mol Sci 14(6):11963–11980.  https://doi.org/10.3390/ijms140611963 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Adams LA, Lymp JF, St Sauver J, Sanderson SO, Lindor KD, Feldstein A, Angulo P (2005) The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 129(1):113–121CrossRefGoogle Scholar
  11. 11.
    Takahashi Y, Soejima Y, Fukusato T (2012) Animal models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. World J Gastroenterol 18(19):2300–2308.  https://doi.org/10.3748/wjg.v18.i19.2300 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Charlton M, Krishnan A, Viker K, Sanderson S, Cazanave S, McConico A, Masuoko H, Gores G (2011) Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition. Am J Physiol Gastrointest Liver Physiol 301(5):G825–G834.  https://doi.org/10.1152/ajpgi.00145.2011 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sellmann C, Priebs J, Landmann M, Degen C, Engstler AJ, Jin CJ, Garttner S, Spruss A, Huber O, Bergheim I (2015) Diets rich in fructose, fat or fructose and fat alter intestinal barrier function and lead to the development of nonalcoholic fatty liver disease over time. J Nutr Biochem 26(11):1183–1192.  https://doi.org/10.1016/j.jnutbio.2015.05.011 CrossRefPubMedGoogle Scholar
  14. 14.
    Tipoe GL, Ho CT, Liong EC, Leung TM, Lau TY, Fung ML, Nanji AA (2009) Voluntary oral feeding of rats not requiring a very high fat diet is a clinically relevant animal model of non-alcoholic fatty liver disease (NAFLD). Histol Histopathol 24(9):1161–1169.  https://doi.org/10.14670/HH-24.1161 CrossRefPubMedGoogle Scholar
  15. 15.
    Green CJ, Pramfalk C, Morten KJ, Hodson L (2015) From whole body to cellular models of hepatic triglyceride metabolism: man has got to know his limitations. Am J Physiol Endocrinol Metab 308(1):E1–E20.  https://doi.org/10.1152/ajpendo.00192.2014 CrossRefPubMedGoogle Scholar
  16. 16.
    Chavez-Tapia NC, Rosso N, Tiribelli C (2012) Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease. BMC Gastroenterol 12:20.  https://doi.org/10.1186/1471-230X-12-20 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Gomez-Lechon MJ, Donato MT, Martinez-Romero A, Jimenez N, Castell JV, O’Connor JE (2007) A human hepatocellular in vitro model to investigate steatosis. Chem Biol Interact 165(2):106–116.  https://doi.org/10.1016/j.cbi.2006.11.004 CrossRefPubMedGoogle Scholar
  18. 18.
    Hirahatake KM, Meissen JK, Fiehn O, Adams SH (2011) Comparative effects of fructose and glucose on lipogenic gene expression and intermediary metabolism in HepG2 liver cells. PLoS One 6(11):e26583.  https://doi.org/10.1371/journal.pone.0026583 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Ricchi M, Odoardi MR, Carulli L, Anzivino C, Ballestri S, Pinetti A, Fantoni LI, Marra F, Bertolotti M, Banni S, Lonardo A, Carulli N, Loria P (2009) Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. J Gastroenterol Hepatol 24(5):830–840.  https://doi.org/10.1111/j.1440-1746.2008.05733.x CrossRefPubMedGoogle Scholar
  20. 20.
    Ishii M, Maeda A, Tani S, Akagawa M (2015) Palmitate induces insulin resistance in human HepG2 hepatocytes by enhancing ubiquitination and proteasomal degradation of key insulin signaling molecules. Arch Biochem Biophys 566:26–35.  https://doi.org/10.1016/j.abb.2014.12.009 CrossRefPubMedGoogle Scholar
  21. 21.
    Rajalin AM, Micoogullari M, Sies H, Steinbrenner H (2014) Upregulation of the thioredoxin-dependent redox system during differentiation of 3T3-L1 cells to adipocytes. Biol Chem 395(6):667–677.  https://doi.org/10.1515/hsz-2014-0102 CrossRefPubMedGoogle Scholar
  22. 22.
    Urban N, Tsitsipatis D, Hausig F, Kreuzer K, Erler K, Stein V, Ristow M, Steinbrenner H, Klotz LO (2017) Non-linear impact of glutathione depletion on C. elegans life span and stress resistance. Redox Biol 11:502–515.  https://doi.org/10.1016/j.redox.2016.12.003 CrossRefPubMedGoogle Scholar
  23. 23.
    Speckmann B, Walter PL, Alili L, Reinehr R, Sies H, Klotz LO, Steinbrenner H (2008) Selenoprotein P expression is controlled through interaction of the coactivator PGC-1alpha with FoxO1a and hepatocyte nuclear factor 4alpha transcription factors. Hepatology 48(6):1998–2006.  https://doi.org/10.1002/hep.22526 CrossRefPubMedGoogle Scholar
  24. 24.
    Schmolz L, Schubert M, Kirschner J, Kluge S, Galli F, Birringer M, Wallert M, Lorkowski S (2018) Long-chain metabolites of vitamin E: Interference with lipotoxicity via lipid droplet associated protein PLIN2. Biochim Biophys Acta Mol Cell Biol Lipids 1863(8):919–927.  https://doi.org/10.1016/j.bbalip.2018.05.002 CrossRefPubMedGoogle Scholar
  25. 25.
    Fosang AJ, Colbran RJ (2015) Transparency is the key to quality. J Biol Chem 290(50):29692–29694.  https://doi.org/10.1074/jbc.E115.000002 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414(6865):813–820.  https://doi.org/10.1038/414813a CrossRefPubMedGoogle Scholar
  27. 27.
    Sage AT, Walter LA, Shi Y, Khan MI, Kaneto H, Capretta A, Werstuck GH (2010) Hexosamine biosynthesis pathway flux promotes endoplasmic reticulum stress, lipid accumulation, and inflammatory gene expression in hepatic cells. Am J Physiol Endocrinol Metab 298(3):E499–E511.  https://doi.org/10.1152/ajpendo.00507.2009 CrossRefPubMedGoogle Scholar
  28. 28.
    Straub BK, Stoeffel P, Heid H, Zimbelmann R, Schirmacher P (2008) Differential pattern of lipid droplet-associated proteins and de novo perilipin expression in hepatocyte steatogenesis. Hepatology 47(6):1936–1946.  https://doi.org/10.1002/hep.22268 CrossRefGoogle Scholar
  29. 29.
    Choi CS, Savage DB, Kulkarni A, Yu XX, Liu ZX, Morino K, Kim S, Distefano A, Samuel VT, Neschen S, Zhang D, Wang A, Zhang XM, Kahn M, Cline GW, Pandey SK, Geisler JG, Bhanot S, Monia BP, Shulman GI (2007) Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance. J Biol Chem 282(31):22678–22688.  https://doi.org/10.1074/jbc.M704213200 CrossRefPubMedGoogle Scholar
  30. 30.
    Yu XX, Murray SF, Pandey SK, Booten SL, Bao D, Song XZ, Kelly S, Chen S, McKay R, Monia BP, Bhanot S (2005) Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice. Hepatology 42(2):362–371.  https://doi.org/10.1002/hep.20783 CrossRefPubMedGoogle Scholar
  31. 31.
    Graffmann N, Ring S, Kawala MA, Wruck W, Ncube A, Trompeter HI, Adjaye J (2016) Modeling nonalcoholic fatty liver disease with human pluripotent stem cell-derived immature hepatocyte-like cells reveals activation of PLIN2 and confirms regulatory functions of peroxisome proliferator-activated receptor alpha. Stem Cells Dev 25(15):1119–1133.  https://doi.org/10.1089/scd.2015.0383 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Green H, Meuth M (1974) An established pre-adipose cell line and its differentiation in culture. Cell 3(2):127–133CrossRefGoogle Scholar
  33. 33.
    Lambert JE, Ramos-Roman MA, Browning JD, Parks EJ (2014) Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease. Gastroenterology 146(3):726–735.  https://doi.org/10.1053/j.gastro.2013.11.049 CrossRefGoogle Scholar
  34. 34.
    Do MT, Kim HG, Choi JH, Khanal T, Park BH, Tran TP, Hwang YP, Na M, Jeong HG (2013) Phillyrin attenuates high glucose-induced lipid accumulation in human HepG2 hepatocytes through the activation of LKB1/AMP-activated protein kinase-dependent signalling. Food Chem 136(2):415–425.  https://doi.org/10.1016/j.foodchem.2012.09.012 CrossRefPubMedGoogle Scholar
  35. 35.
    Li H, Min Q, Ouyang C, Lee J, He C, Zou MH, Xie Z (2014) AMPK activation prevents excess nutrient-induced hepatic lipid accumulation by inhibiting mTORC1 signaling and endoplasmic reticulum stress response. Biochim Biophys Acta 1842(9):1844–1854.  https://doi.org/10.1016/j.bbadis.2014.07.002 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Zhang Y, Takemori H, Wang C, Fu J, Xu M, Xiong L, Li N, Wen X (2017) Role of salt inducible kinase 1 in high glucose-induced lipid accumulation in HepG2 cells and metformin intervention. Life Sci 173:107–115.  https://doi.org/10.1016/j.lfs.2017.02.001 CrossRefPubMedGoogle Scholar
  37. 37.
    Li S, Brown MS, Goldstein JL (2010) Bifurcation of insulin signaling pathway in rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of gluconeogenesis. Proc Natl Acad Sci USA 107(8):3441–3446.  https://doi.org/10.1073/pnas.0914798107 CrossRefPubMedGoogle Scholar
  38. 38.
    Bartolini D, Torquato P, Barola C, Russo A, Rychlicki C, Giusepponi D, Bellezza G, Sidoni A, Galarini R, Svegliati-Baroni G, Galli F (2017) Nonalcoholic fatty liver disease impairs the cytochrome P-450-dependent metabolism of alpha-tocopherol (vitamin E). J Nutr Biochem 47:120–131.  https://doi.org/10.1016/j.jnutbio.2017.06.003 CrossRefPubMedGoogle Scholar
  39. 39.
    Zhao L, Guo X, Wang O, Zhang H, Wang Y, Zhou F, Liu J, Ji B (2016) Fructose and glucose combined with free fatty acids induce metabolic disorders in HepG2 cell: a new model to study the impacts of high-fructose/sucrose and high-fat diets in vitro. Mol Nutr Food Res 60(4):909–921.  https://doi.org/10.1002/mnfr.201500635 CrossRefPubMedGoogle Scholar
  40. 40.
    Meissen JK, Hirahatake KM, Adams SH, Fiehn O (2015) Temporal metabolomic responses of cultured HepG2 liver cells to high fructose and high glucose exposures. Metabolomics 11(3):707–721.  https://doi.org/10.1007/s11306-014-0729-8 CrossRefPubMedGoogle Scholar
  41. 41.
    Beriault DR, Sharma S, Shi Y, Khan MI, Werstuck GH (2011) Glucosamine-supplementation promotes endoplasmic reticulum stress, hepatic steatosis and accelerated atherogenesis in apoE−/− mice. Atherosclerosis 219(1):134–140.  https://doi.org/10.1016/j.atherosclerosis.2011.07.108 CrossRefPubMedGoogle Scholar
  42. 42.
    Sztalryd C, Kimmel AR (2014) Perilipins: lipid droplet coat proteins adapted for tissue-specific energy storage and utilization, and lipid cytoprotection. Biochimie 96:96–101.  https://doi.org/10.1016/j.biochi.2013.08.026 CrossRefPubMedGoogle Scholar
  43. 43.
    Pawella LM, Hashani M, Eiteneuer E, Renner M, Bartenschlager R, Schirmacher P, Straub BK (2014) Perilipin discerns chronic from acute hepatocellular steatosis. J Hepatol 60(3):633–642.  https://doi.org/10.1016/j.jhep.2013.11.007 CrossRefPubMedGoogle Scholar
  44. 44.
    Masuda Y, Itabe H, Odaki M, Hama K, Fujimoto Y, Mori M, Sasabe N, Aoki J, Arai H, Takano T (2006) ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells. J Lipid Res 47(1):87–98.  https://doi.org/10.1194/jlr.M500170-JLR200 CrossRefGoogle Scholar
  45. 45.
    Kaushik S, Cuervo AM (2015) Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis. Nat Cell Biol 17(6):759–770.  https://doi.org/10.1038/ncb3166 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Cases S, Stone SJ, Zhou P, Yen E, Tow B, Lardizabal KD, Voelker T, Farese RV Jr (2001) Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members. J Biol Chem 276(42):38870–38876.  https://doi.org/10.1074/jbc.M106219200 CrossRefPubMedGoogle Scholar
  47. 47.
    Meegalla RL, Billheimer JT, Cheng D (2002) Concerted elevation of acyl-coenzyme A: diacylglycerol acyltransferase (DGAT) activity through independent stimulation of mRNA expression of DGAT1 and DGAT2 by carbohydrate and insulin. Biochem Biophys Res Commun 298(3):317–323CrossRefGoogle Scholar
  48. 48.
    Wang S, Wang J, Zhang X, Hu L, Fang Z, Huang Z, Shi P (2016) Trivalent chromium alleviates oleic acid induced steatosis in SMMC-7721 cells by decreasing fatty acid uptake and triglyceride synthesis. Biometals 29(5):881–892.  https://doi.org/10.1007/s10534-016-9960-2 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Nutritional Sciences, NutrigenomicsFriedrich-Schiller-Universität JenaJenaGermany
  2. 2.Department of Nutritional Biochemistry and Physiology, Institute of Nutritional SciencesFriedrich-Schiller-Universität JenaJenaGermany
  3. 3.Competence Cluster for Nutrition and Cardiovascular Health (nutriCARD) Halle-Jena-LeipzigJenaGermany

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