Advertisement

Lack of liver steatosis in germ-free mice following hypercaloric diets

  • Valentina Kaden-Volynets
  • Marijana Basic
  • Ulrike Neumann
  • Dominik Pretz
  • Andreas Rings
  • André Bleich
  • Stephan C. Bischoff
Original Contribution

Abstract

Purpose

Experimental liver steatosis induced by overfeeding is associated with enhanced gut permeability and endotoxin translocation to the liver. We examined the role of the gut microbiota for steatosis formation by performing the feeding experiments in mice raised under conventional and germ-free (GF) housing.

Methods

Adult wild-type and GF mice were fed a Western-style diet (WSD) or a control diet (CD), the latter combined with liquid fructose supplementation (F) or not, for 8 weeks. Markers of liver steatosis and gut permeability were measured after intervention.

Results

Mice fed a WSD increased body weight compared to those fed a CD (p < 0.01) under conventional, but not under GF conditions. Increased liver weight, liver-to-body-weight ratio and hepatic triglycerides observed in both the WSD and the CD + F groups, when compared with the CD group, were not apparent under GF conditions, whereas elevated plasma triglycerides were visible (p < 0.05). Wild-type mice fed a WSD or a CD + F, respectively, had thinner adherent mucus layer compared to those fed a CD (p < 0.01), whereas GF mice had always a thin mucus layer independently of the diet. GF mice fed a CD showed increased plasma levels of FITC-dextran 4000 (1.9-fold, p < 0.05) and intestinal fatty acid-binding protein-2 (2.4-fold, p < 0.05) compared with wild-type mice.

Conclusions

GF housing results in an impaired weight gain and a lack of steatosis following a WSD. Also the fructose-induced steatosis, which is unrelated to body weight changes, is absent in GF mice. Thus, diet-induced experimental liver steatosis depends in multiple ways on intestinal bacteria.

Keywords

Germ free Fructose Intestinal barrier Mucin Liver steatosis Western-style diet 

Notes

Acknowledgements

Supported in part by a Grant from the German Research Foundation (BI 424/8-1; to SCB).

Author contributions

SCB and AB conception and design of the study; SCB funding of the study; VV and MB conduction of the studies, AR, DP, UN and VV acquisition of data; SCB and VV statistical analysis and interpretation of data; AR technical and material support; AB and MB provided the germ-free facility; SCB and VV drafting the manuscript; AB and MB revising the manuscript critically for important intellectual content; SCB had primary responsibility for final content.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Cusi K (2012) Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: pathophysiology and clinical implications. Gastroenterology 142:711–725.e6CrossRefPubMedGoogle Scholar
  2. 2.
    Yki-Järvinen H (2014) Non-alcoholic fatty liver disease as a cause and a consequence of metabolic syndrome. Lancet Diabetes Endocrinol 2:901–910CrossRefPubMedGoogle Scholar
  3. 3.
    Machado MV, Cortez-Pinto H (2011) No need for a large belly to have NASH. J Hepatol 54:1090–1093CrossRefPubMedGoogle Scholar
  4. 4.
    Stanton MC, Chen SC, Jackson JV, Rojas-Triana A, Kinsley D, Cui L, Fine JS, Greenfeder S, Bober LA, Jenh CH (2011) Inflammatory signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice. J Inflamm (Lond) 8:8CrossRefGoogle Scholar
  5. 5.
    Volynets V, Louis S, Pretz D, Lang L, Ostaff MJ, Wehkamp J, Bischoff SC (2017) Intestinal barrier function and the gut microbiome are differentially affected in mice fed a western-style diet or drinking water supplemented with fructose. J Nutr 147:770–780CrossRefPubMedGoogle Scholar
  6. 6.
    Spruss A, Kanuri G, Stahl C, Bischoff SC, Bergheim I (2012) Metformin protects against the development of fructose-induced steatosis in mice: role of the intestinal barrier function. Lab Invest 92:1020–1032CrossRefPubMedGoogle Scholar
  7. 7.
    Reichold A, Brenner SA, Spruss A, Förster-Fromme K, Bergheim I, Bischoff SC (2014) Bifidobacterium adolescentis protects from the development of nonalcoholic steatohepatitis in a mouse model. J Nutr Biochem 25:118–125CrossRefGoogle Scholar
  8. 8.
    Spruss A, Kanuri G, Wagnerberger S, Haub S, Bischoff SC, Bergheim I (2009) Toll-like receptor 4 is involved in the development of fructose-induced hepatic steatosis in mice. Hepatology 50:1094–1104CrossRefGoogle Scholar
  9. 9.
    Volynets V, Reichold A, Bárdos G, Rings A, Bleich A, Bischoff SC (2016) Assessment of the intestinal barrier with five different permeability tests in healthy C57BL/6J and BALB/cJ mice. Dig Dis Sci 61:737–746CrossRefPubMedGoogle Scholar
  10. 10.
    Schroyen M, Stinckens A, Verhelst R, Janssens S, Niewold T, Buys N (2011) IFABP expression as diagnostic tool for integrity of epithelium. Commun Agric Appl Biol Sci 76:53–56PubMedGoogle Scholar
  11. 11.
    Johansson ME, Hansson GC (2012) Preservation of mucus in histological sections, immunostaining of mucins in fixed tissue, and localization of bacteria with FISH. Methods Mol Biol 842:229–235CrossRefPubMedGoogle Scholar
  12. 12.
    Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925PubMedPubMedCentralGoogle Scholar
  13. 13.
    Wehkamp J, Wang G, Kübler I, Nuding S, Gregorieff A, Schnabel A, Kays RJ, Fellermann K, Burk O, Schwab M, Clevers H, Bevins CL, Stange EF (2007) The Paneth cell alpha-defensin deficiency of ileal Crohn’s disease is linked to Wnt/Tcf-4. J Immunol 179:3109–3118CrossRefPubMedGoogle Scholar
  14. 14.
    Ferré P, Foufelle F (2010) Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab 2:83–92CrossRefGoogle Scholar
  15. 15.
    Johansson ME, Ambort D, Pelaseyed T, Schütte A, Gustafsson JK, Ermund A, Subramani DB, Holmén-Larsson JM, Thomsson KA, Bergström JH, van der Post S, Rodriguez-Piñeiro AM, Sjövall H, Bäckström M, Hansson GC (2011) Composition and functional role of the mucus layers in the intestine. Cell Mol Life Sci 68:3635–3641CrossRefPubMedGoogle Scholar
  16. 16.
    Madsen J, Nielsen O, Tornøe I, Thim L, Holmskov U (2007) Tissue localization of human trefoil factors 1, 2, and 3. J Histochem Cytochem 55:505–513CrossRefPubMedGoogle Scholar
  17. 17.
    Bergheim I, Weber S, Vos M, Krämer S, Volynets V, Kaserouni S, McClain CJ, Bischoff SC (2008) Antibiotics protect against fructose-induced hepatic lipid accumulation in mice: role of endotoxin. J Hepatol 48:983–992CrossRefGoogle Scholar
  18. 18.
    Tremaroli V, Backhed F (2012) Functional interactions between the gut microbiota and host metabolism. Nature 489:242–249CrossRefGoogle Scholar
  19. 19.
    Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, Semenkovich CF, Gordon JI (2004) The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 101:15718–15723CrossRefGoogle Scholar
  20. 20.
    Gordon HA, Pesti L (1972) The gnotobiotic animal as a tool in the study of host microbial relationships. Bacteriol Rev 35:390–429Google Scholar
  21. 21.
    Gustafsson BE, Carlstedt-Duke B (1984) Intestinal water-soluble mucins in germfree, exgermfree and conventional animals. Acta Pathol Microbiol Immunol Scand 92:247–252Google Scholar
  22. 22.
    Fukushima K, Sasaki I, Ogawa H, Naito H, Funayama Y, Matsuno S (1999) Colonization of microflora in mice: mucosal defense against luminal bacteria. J Gastroenterol 34:54–60CrossRefPubMedGoogle Scholar
  23. 23.
    Damms-Machado A, Louis S, Schnitzer A, Volynets V, Rings A, Basrai M, Bischoff SC (2017) Gut permeability is related to body weight, fatty liver disease, and insulin resistance in obese individuals undergoing weight reduction. Am J Clin Nutr 105:127–135CrossRefPubMedGoogle Scholar
  24. 24.
    Ridaura VK, Faith JJ, Rey FE, Cheng J, Duncan AE, Kau AL, Griffin NW, Lombard V, Henrissat B, Bain JR, Muehlbauer MJ, Ilkayeva O, Semenkovich CF, Funai K, Hayashi DK, Lyle BJ, Martini MC, Ursell LK, Clemente JC, Van Treuren W, Walters WA, Knight R, Newgard CB, Heath AC, Gordon JI (2013) Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 341:1241214CrossRefGoogle Scholar
  25. 25.
    Lynch SV, Pedersen O (2016) The human intestinal microbiome in health and disease. N Engl J Med 375:2369–2379CrossRefPubMedGoogle Scholar
  26. 26.
    Tilg H, Moschen AR (2014) Microbiota and diabetes: an evolving relationship. Gut 63:1513–1521CrossRefPubMedGoogle Scholar
  27. 27.
    Duparc T, Plovier H, Marrachelli VG, Van Hul M, Essaghir A, Ståhlman M, Matamoros S, Geurts L, Pardo-Tendero MM, Druart C, Delzenne NM, Demoulin JB, van der Merwe SW, van Pelt J, Bäckhed F, Monleon D, Everard A, Cani PD (2017) Hepatocyte MyD88 affects bile acids, gut microbiota and metabolome contributing to regulate glucose and lipid metabolism. Gut 66:620–632CrossRefPubMedGoogle Scholar
  28. 28.
    Geurts L, Neyrinck AM, Delzenne NM, Knauf C, Cani PD (2014) Gut microbiota controls adipose tissue expansion, gut barrier and glucose metabolism: novel insights into molecular targets and interventions using prebiotics. Benef Microbes 5:3–17CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Valentina Kaden-Volynets
    • 1
  • Marijana Basic
    • 2
  • Ulrike Neumann
    • 1
  • Dominik Pretz
    • 1
    • 3
  • Andreas Rings
    • 1
  • André Bleich
    • 2
  • Stephan C. Bischoff
    • 1
  1. 1.Department of Nutritional MedicineUniversity of HohenheimStuttgartGermany
  2. 2.Institute for Laboratory Animal Science, Hannover Medical SchoolHannoverGermany
  3. 3.Department of Physiology, Otago School of Medical SciencesUniversity of OtagoDunedinNew Zealand

Personalised recommendations