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Three-dimensional culture model for analyzing crosstalk between adipose tissue and hepatocytes

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Abstract

Systemic adipose tissue is involved in the pathophysiology of obesity-associated liver diseases. However, a method has not been established for analyzing the direct interaction between adipose tissue and hepatocytes. We describe a useful three-dimensional model comprising a collagen gel coculture system in which HepG2 hepatocytes are cultured on a gel layer with visceral adipose tissue fragments (VAT) or subcutaneous tissue samples (SAT). Male adipose tissues were obtained from 5-week-old Wistar rats and human autopsy cases. Cellular behavior was analyzed by electron microscopy, immunohistochemistry, Western blot, real-time reverse transcription plus the polymerase chain reaction and enzyme-linked immunosorbent assay. VAT significantly promoted lipid accumulation and apoptosis in HepG2 cells and suppressed their growth and differentiation compared with SAT. VAT produced higher concentrations of fatty acids (palmitate, oleate, linoleate) than SAT. HepG2 cells significantly decreased the production of these fatty acids in VAT. Only HepG2 cells treated with 250 μM palmitate replicated VAT-induced apoptosis. Neither VAT nor SAT affected lipotoxicity-associated signals of nuclear factor kappa B, c-Jun N-terminal kinase and inositol requiring enzyme-1α in HepG2 cells. HepG2 cells never affected adiponectin, leptin, or resistin production in VAT and SAT. The data indicate that our model actively creates adipose tissue and HepG2 hepatocyte interactions, suggesting that (1) VAT plays more critical roles in hepatocyte lipotoxicity than SAT; (2) palmitate but not adipokines, is partly involved in the mechanisms of VAT-induced lipotoxicity; (3) HepG2 cells might inhibit fatty acid production in VAT to protect themselves against lipotoxicity. Our model should serve in studies of interactions between adipose tissue and hepatocytes and of the mechanisms in obesity-related lipotoxicity and liver diseases.

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Acknowledgments

We are grateful to Dr. S. Ohta for his excellent suggestions and to H. Ideguchi, S. Nakahara, F. Mutoh and M. Nishida for their excellent technical assistance.

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Authors and Affiliations

  1. Department of Pathology & Microbiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga, 849-8501, Japan

    Aki Nishijima-Matsunobu, Shigehisa Aoki, Kazuyoshi Uchihashi & Shuji Toda

  2. Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga, 849-8501, Japan

    Aki Nishijima-Matsunobu & Kazuma Fujimoto

Authors
  1. Aki Nishijima-Matsunobu
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  2. Shigehisa Aoki
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  3. Kazuyoshi Uchihashi
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  4. Kazuma Fujimoto
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  5. Shuji Toda
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Corresponding author

Correspondence to Aki Nishijima-Matsunobu.

Additional information

This work was supported in part by Japanese Ministry of Education, Culture, Sports, Science and Technology Grants-in-Aid for Scientific Research nos. 22590740 (to A.N.-M.), 18591871 and 20592023 (to S.T.) and personal grants from Koike Hospital, Sasebo Chuo Hospital and Yamada Clinic (to S.T.).

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Fig. S1

3T3 fibroblasts never induce the rat and human VAT-affected morphology of the cells. Scale bars = 50 μm. (JPEG 11 kb)

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Fig. S2

Lipid accumulation of RL-34 cells cultured with rat adipose tissues are detected in red by oil red O stain. a RL-34 cells alone have no lipid droplets. b, c Culture with rVAT (c) promotes more prominent lipid deposition in RL-34 cells than culture with rSAT (b). (JPEG 19 kb)

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Fig. S3

Fatty acid analyses in rSAT and rVAT. In fresh VAT and SAT just after resection from rats, large amounts of palmitate (C16:0), oleate (C18:1ω9) and linoleate (C18:2ω6) are detected. The contents of these fatty acids are higher in SAT than in VAT. *P < 0.05, **P < 0.01, ***P < 0.001. (JPEG 30 kb)

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Nishijima-Matsunobu, A., Aoki, S., Uchihashi, K. et al. Three-dimensional culture model for analyzing crosstalk between adipose tissue and hepatocytes. Cell Tissue Res 352, 611–621 (2013). https://doi.org/10.1007/s00441-013-1588-8

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  • DOI: https://doi.org/10.1007/s00441-013-1588-8

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