An effective protocol for pharmacological defatting of primary human hepatocytes which is non-toxic to cholangiocytes or intrahepatic endothelial cells

Abstract

Introduction: Pharmacological defatting of rat hepatocytes and hepatoma cell lines suggests that the same method could be used to ameliorate macrovesicular steatosis in moderate to severely fatty livers. However there is no data assessing the effects of those drugs on primary human liver cells. We aimed to determine the effectiveness of a pharmacological cocktail in reducing the in vitro lipid content of primary human hepatocytes (PHH). In addition we sought to determine the cytotoxicity of the cocktail towards non-parenchymal liver cells.

Methods: Steatosis was induced in PHH by supplementation with a combination of saturated and unsaturated free fatty acids. This was followed by addition of a defatting drug cocktail for up to 48 hours. The same experimental method was used with human intra-hepatic endothelial cells (HIEC) and human cholangiocytes. MTT assay was used to assess cell viability, triglyceride quantification and oil red O staining were used to determine intracellular lipids content whilst ketone bodies were measured in the supernatants following experimentation.

Results: Incubation of fat loaded PHH with the drugs over 48 hours reduced the intracellular lipid area by 54%, from 12.85% to 5.99% (p = 0.002) (percentage of total oil red O area), and intracellular triglyceride by 35%, from 28.24 to 18.30 nmol/million of cells (p<0.001). Total supernatant ketone bodies increased 1.4-fold over 48 hours in the defatted PHH compared with vehicle controls (p = 0.002). Moreover incubation with the drugs for 48 hours increased the viability of PHH by 11%, cholangiocytes by 25% whilst having no cytotoxic effects on HIEC.

Conclusion: These data demonstrate that pharmacological intervention can significantly decrease intracellular lipid content of PHH, increase fatty acids β-oxidation whilst being non-toxic to PHH, HIEC or cholangiocytes.

Fig 1. Study design.

Series 1: Isolated primary human hepatocytes (PHH) were left in standard media for 2 days and then received media supplemented with fatty acids. After 2 days of fat loading the fatty PHH were allocated to the defatting treatment group where the media was supplemented with the defatting cocktail of drugs, and the control groups, the standard control group and the vehicle control group that received vehicle only. Lean hepatocytes were kept in standard culture conditions throughout the experimental period. The experimentation period lasted for two days thereafter. Series 2: Human intra-hepatic endothelial cells (HIEC) and cholangiocytes were immuno-magnetically separated with Dynabeads conjugated with cell-specific monoclonal antibody. The cells were left in culture for 2 days in standard media to reach confluence and then were allocated to the intervention group that received the defatting cocktail and the control groups, the standard control group and the vehicle control group that had the media supplemented with the vehicle only. The experimentation period lasts for two days thereafter.

Fig 2. Results of fat loading of primary human hepatocytes.

Panel A: The supplementation of the media with the combination of fatty acids resulted in a cell viability rate of 81% after 48 hours of incubation. Panel B: Oil red O staining image of PHH at the end of the fat loading period. There is predominance of large lipid droplets displacing the nucleus of the cells to the periphery (black arrow). Panel C: At the end of 48 hours of fatting load there was a significant increase of 14-fold of the positive area of oil red O. Panel D: Intracellular triglycerides increased 8-fold within 48 hours of incubation with fatty acids. Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.

Fig 3. Defatting of fat loaded primary human hepatocytes (PHH).

Panel A: The positive are of oil red O of the defatting treatment group was reduced by 28% in comparison with the vehicle control group over 24 hours and 54% over 48 hours. Panel B: Intracellular triglyceride levels of the defatting treatment group were reduced by 32% within 24 hours of treatment and 35% within 48 hours, in comparison with the fatty vehicle control group. Panel C: Oil red O staining picture of PHH of the defatting group at the end of the 48 hours of treatment. There is a predominance of small lipid droplets in the cytoplasm of the cells and the nucleus is in its usual position. Series 2: shows a series of oil red O staining pictures from PHH in culture at different time points of the experiments. Panel D shows lean cells in culture, after the incubation with fatty acids they become loaded with fat (panel E). Those fat loaded PHH were then incubated with only the vehicle of the drugs for 48 hours and the lipid content decreased over time (panel F) or had the defatting treatment that showed the significant higher decrease in the area of oil red O (panel G). Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.

Fig 4. Release of total ketones in the supernatants.

Fat loaded primary human hepatocytes that had the defatting treatment showed an increase in cell culture supernatant levels of total ketone bodies of 1.22-fold over 24 hours and 1.40-fold over 48 hours. Data reports the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.

Fig 5. Assessment of the cytotoxicity of the defatting cocktail to human cells of the liver via MTT assay.

Panel A: the toxicity of the defatting cocktail was tested in primary human hepatocytes and results showed a significant improvement of 11% in viability of the defatting treatment group compared with the fatty vehicle control group. Panel B: Treatment of human intra-hepatic endothelial cells (HIEC) with the drugs had no effect on cell viability compared with the control groups. Panel C: treatment of cholangiocytes with the defatting cocktail did not demonstrate any cytotoxic effect to the cells and indicated a slight improvement in viability compared to the control groups. Panels D and E: Phase contrast microscopy showing representative images of HIEC (Panel D) and cholangiocytes (Panel E) at different time points. No gross modifications in cell integrity were observed in either cell type which was consistent and supportive of the MTT data. Data report the median of three separate experiments performed in quadruplicate and errors bars the interquartile range. Comparisons performed using two-tailed t-test. * = p<0.05.