Top

European Journal of Nutrition

Published in:

01-12-2019 | Original Contribution

Differential capability of metabolic substrates to promote hepatocellular lipid accumulation

Authors: Ngoc Anh Hoang, Friederike Richter, Martin Schubert, Stefan Lorkowski, Lars-Oliver Klotz, Holger Steinbrenner

Published in: European Journal of Nutrition | Issue 8/2019

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.

Available only for authorised users

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

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):113–121CrossRef

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

26.

Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414(6865):813–820. https://doi.org/10.1038/414813a CrossRefPubMed

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 CrossRefPubMed

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 CrossRef

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

32.

Green H, Meuth M (1974) An established pre-adipose cell line and its differentiation in culture. Cell 3(2):127–133CrossRef

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 CrossRef

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 CrossRefPubMed

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRefPubMed

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 CrossRef

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 CrossRefPubMedPubMedCentral

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 CrossRefPubMed

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):317–323CrossRef

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 CrossRefPubMed

Title: Differential capability of metabolic substrates to promote hepatocellular lipid accumulation
Authors: Ngoc Anh Hoang
Friederike Richter
Martin Schubert
Stefan Lorkowski
Lars-Oliver Klotz
Holger Steinbrenner
Publication date: 01-12-2019
Publisher: Springer Berlin Heidelberg
Published in: European Journal of Nutrition / Issue 8/2019
Print ISSN: 1436-6207
Electronic ISSN: 1436-6215
DOI: https://doi.org/10.1007/s00394-018-1847-2

Live Webinar | 27-06-2024 | 18:00 (CEST)

Keynote webinar | Spotlight on medication adherence

Reserve your place

Live: Thursday 27th June 2024, 18:00-19:30 (CEST)

WHO estimates that half of all patients worldwide are non-adherent to their prescribed medication. The consequences of poor adherence can be catastrophic, on both the individual and population level.

Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.

Prof. Kevin Dolgin

Prof. Florian Limbourg

Prof. Anoop Chauhan

Developed by: Springer Medicine

Obesity Clinical Trial Summary

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.

Developed by: Springer Medicine

Highlights from the ACC 2024 Congress

Year in Review: Pediatric cardiology

Pediatric Cardiology Video

Watch Dr. Anne Marie Valente present the last year's highlights in pediatric and congenital heart disease in the official ACC.24 Year in Review session.

Year in Review: Pulmonary vascular disease

Cardiopulmonary Comorbidity Video

The last year's highlights in pulmonary vascular disease are presented by Dr. Jane Leopold in this official video from ACC.24.

Year in Review: Valvular heart disease

Valvular Heart Disease Video

Watch Prof. William Zoghbi present the last year's highlights in valvular heart disease from the official ACC.24 Year in Review session.

Year in Review: Heart failure and cardiomyopathies

Heart Failure Video

Watch this official video from ACC.24. Dr. Biykem Bozkurt discusses last year's major advances in heart failure and cardiomyopathies.

ACC 2024 Congress Coverage

Year in Review: Heart failure and cardiomyopathies

Heart Failure Video

Watch this official video from ACC.24. Dr. Biykem Bozkurt discusses last year's major advances in heart failure and cardiomyopathies.

Watch now

Semaglutide benefits people with type 2 diabetes and obesity-related heart failure

16-04-2024 Type 2 Diabetes News

Incentivised to exercise

11-04-2024 Lifestyle Modifications News

New intervention for STEMI-related cardiogenic shock

10-04-2024 Myocardial Infarction News

» View more highlights on the ACC 2024 congress hub page

Image Credits

Illustration of figure on mountain summit/© Orapun / Stock.adobe.com, STEP AAG HeroTeaserCollection image/© Tarek / Generated with AI / Stock.adobe.com, ACC 2024 Pediatric and Congenital Heart Disease: Year in Review/© 2024 American College of Cardiology, ACC 2024 Pulmonary Vascular Disease: Year in Review/© 2024 American College of Cardiology, ACC 2024 Valvular Heart Disease: Year in Review/© 2024 American College of Cardiology, ACC 2024 HF and Cardiomyopathies: Year in Review/© 2024 American College of Cardiology, Woman out walking/© Seventyfour / stock.adobe.com (symbolic image with model), Woman checking smartwatch /© saltdium / stock.adobe.com (symbolic image with model), Cardiac catheterization/© Damian / stock.adobe.com

At a glance: The STEP trials

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 8/2019

N-Acetylcysteine protects against intrauterine growth retardation-induced intestinal injury via restoring redox status and mitochondrial function in neonatal piglets

Soy and isoflavones consumption and breast cancer survival and recurrence: a systematic review and meta-analysis

Correction to: The novel insight into anti-inflammatory and anxiolytic effects of psychobiotics in diabetic rats: possible link between gut microbiota and brain regions

Association between dietary cadmium intake and early gastric cancer risk in a Korean population: a case–control study

Rice intake and risk of type 2 diabetes: the Singapore Chinese Health Study

A proteomics–metabolomics approach indicates changes in hypothalamic glutamate–GABA metabolism of adult female rats submitted to intrauterine growth restriction

Live: Thursday 27th June 2024, 18:00-19:30 (CEST)

Highlights from the ACC 2024 Congress

ACC 2024 Congress Coverage