An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro

Silke Vogel,Roseann Piantedosi,Jorge Frank,Avraham Lalazar,Don C Rockey,Scott L Friedman,William S Blaner

doi:10.1016/s0022-2275(20)32030-7

Silke Vogel, Roseann Piantedosi + Show 5 more

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https://doi.org/10.1016/s0022-2275(20)32030-7

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Abstract

Hepatocytes and hepatic stellate cells play important roles in retinoid storage and metabolism. Hepatocytes process postprandial retinyl esters and are responsible for secretion of retinol bound to retinol-binding protein (RBP) to maintain plasma retinol levels. Stellate cells are the body's major cellular storage sites for retinoid. We have characterized and utilized an immortalized rat stellate cell line, HSC-T6 cells, to facilitate study of the cellular aspects of hepatic retinoid processing. For comparison, we also carried out parallel studies in Hepa-1 hepatocytes. Like activated primary stellate cells, HSC-T6 express myogenic and neural crest cytoskeletal filaments. HSC-T6 cells take up and esterify retinol in a time- and concentration-dependent manner. Supplementation of HSC-T6 culture medium with free fatty acids (up to 300 μm) does not affect retinol uptake but does enhance retinol esterification up to 10-fold. RT-PCR analysis indicates that HSC-T6 cells express all 6 retinoid nuclear receptors (RARα, -β, -γ, and RXRα, -β, -γ) and like primary stellate cells, HSC-T6 stellate cells express cellular retinol-binding protein, type I (CRBP) but fail to express either retinol-binding protein (RBP) or transthyretin (TTR). Addition of retinol (10−8–10−5m) or all-trans-retinoic acid (10−10–10−6m) rapidly up-regulates CRBP expression. Using RAR-specific agonists and antagonists and an RXR-specific agonist, we show that members of the RAR-receptor family modulate HSC-T6 CRBP expression.Thus, HSC-T6 cells display the same retinoid-related phenotype as primary stellate cells in culture and will be a useful tool for study of hepatic retinoid storage and metabolism.

Highlights

Hepatocytes and hepatic stellate cells play important roles in retinoid storage and metabolism
For estimation of retinol-binding protein (RBP), CRBP, and TTR mRNA levels and to assess the influence of retinoid on the expression of these transcripts in HSC-T6 stellate cells and Hepa-1 hepatocytes, cells were plated and incubated overnight as described above. This media was removed and cells were incubated for 12 h at 37ЊC in the dark with media containing different concentrations of either retinol or all-trans-retinoic acid added to the complete culture media (DMEM and Waymouth for Hepa-1 hepatocytes and HSC-T6 stellate cells, respectively)
Among the most striking features of hepatic stellate cells in vivo are the numerous lipid droplets that are present within the cytoplasm of the cells. These lipid droplets are the major storage sites for retinyl ester in the liver and in vivo both the size and number of lipid droplets depend on dietary vitamin A intake [12]

Summary

Introduction

Hepatocytes and hepatic stellate cells play important roles in retinoid storage and metabolism. For estimation of RBP, CRBP, and TTR mRNA levels and to assess the influence of retinoid on the expression of these transcripts in HSC-T6 stellate cells and Hepa-1 hepatocytes, cells were plated and incubated overnight as described above.

Results

Conclusion