Metabolic preconditioning of donor organs: defatting fatty livers by normothermic perfusion ex vivo

Abstract

Fatty liver is a significant risk factor for liver transplantation, and accounts for nearly half of the livers rejected from the donor pool. We hypothesized that metabolic preconditioning via ex vivo perfusion of the liver graft can reduce fat content and increase post-transplant survival to an acceptable range. We describe a perfusate medium containing agents that promote the defatting of hepatocytes and explanted livers. Defatting agents were screened on cultured hepatocytes made fatty by pre-incubation with fatty acids. The most effective agents were then used on fatty livers. Fatty livers were isolated from obese Zucker rats and normothermically perfused with medium containing a combination of defatting agents. This combination decreased the intracellular lipid content of cultured hepatocytes by 35% over 24h, and of perfused livers by 50% over 3h. Metabolite analysis suggests that the defatting cocktail upregulated both lipid oxidation and export. Furthermore, gene expression analysis for several enzymes and transcription factors involved in fatty acid oxidation and triglyceride clearance were elevated. We conclude that a cocktail of defatting agents can be used to rapidly clear excess lipid storage in fatty livers, thus providing a new means to recondition donor livers deemed unacceptable or marginally acceptable for transplantation.

Figure 1

A) Experimental design of in vitro defatting experiments. B) Schematic of isolated ex vivo fatty liver perfusion setup.

Figure 2

Effect of cocktail of defatting agents on hepatocyte TG content 24 hr after defatting. Primary cultured rat hepatocytes were made steatotic by incubation with fatty acid-supplemented medium for 3 days, and then switched to regular medium supplemented with a cocktail of defatting agents (1 μM GW7, 1 μM HYP, 10 μM SCO, 10 μM FOR, 1 ng/ml VIS, and 1 μM GW5). Vehicle controls represent similar cultures exposed to vehicle control (<0.1% DMSO). Intracellular lipid-specific Nile Red staining of hepatocyte cultures A) at the start of defatting; B-C) after 24 hr of defatting; E-G) after 48 hr of defatting with the cocktail as compared to the vehicle controls. H-I) Quantification of Nile Red staining in hepatocytes by image analysis. *: p<0.05 by one-tailed t-test compared to vehicle control.

Figure 3

Mechanism of defatting of steatotic hepatocytes. Steatotic hepatocytes were incubated in culture medium supplemented with the cocktail of defatting agents, or vehicle control (<0.1% DMSO). A) Total ketone bodies (acetoacetate + β-hydroxybutyrate) secreted after 24 hr of defatting. Data are normalized to the vehicle control. Data shown are the means ± SE of results obtained from two separate isolations and performed in 2 to 5 replicate culture dishes (N = 4 to 10). B) TG secreted after 24 hr of defatting. Data are normalized to vehicle controls. Data shown were obtained from two separate isolations and performed in duplicate cultures (N=4), and expressed as means ± SE. White bars are significantly lower (*, p<0.05) than vehicle control. C) Total and mitochondrial oxygen uptake rates measured after 24 hr of defatting incubation. Data shown are the means ± SE of results obtained from two separate isolations and performed in 2 to 5 replicate culture dishes (N ≥ 4).

Figure 4

Gene expression of lipid metabolic pathways involved during defatting. Steatotic hepatocytes were incubated in culture medium supplemented with the cocktail of defatting agents, or vehicle control (<0.1% DMSO). Fatty hepatocytes treated with defatting agents showed increased expression of genes involved in the pathways of lipid oxidation (ACO, PGC1α, CPT1α, PPARα, and PPARδ), lipid secretion (ApoB-100), and lipid hydrolysis (TGH), compared to the vehicle control. The increased expression was observed for defatting durations of both 24 hrs and 48 hrs.

Figure 5

Defatting of steatotic livers isolated from obese fa/fa Zucker rats. Isolated rat livers were perfused at 37°C with perfusate containing the defatting agent cocktail (N=7) or perfusate with DMSO vehicle (N=5) for 3 hr. A) TG content remaining after perfusion. Data are normalized and compared to unperfused freshly isolated steatotic livers (138 mg TG/g liver). B) Total bile production by perfused livers. *: p<0.05 by one-tailed t-test compared to vehicle control.

Figure 6

Histological appearance of defatted steatotic livers. Livers (14-15 weeks of Zucker rats) were harvested before and after 3 hr of perfusion, fixed and stained with hematoxylin and eosin. A) Normal lean liver. B) Fatty liver before perfusion. C) Fatty liver after perfusion with the vehicle control. D) Fatty liver after perfusion with the defatting cocktail. Higher magnification images are shown in right panel.

Figure 7

Mechanism of defatting during perfusion of steatotic livers. A) VLDL secreted into the perfusate after 2 hr of perfusion with the defatting cocktail (N=4) as compared to vehicle controls (N=3). B) Total oxygen uptake rate during fatty liver perfusion with defatting cocktail (N=4) as compared to vehicle control (N=5). C) Total ketone body secretion during fatty liver perfusion with defatting cocktail (N=3) as compared to vehicle control (N=3). Data shown are expressed as means ± SE. *: p<0.05, **: p<0.01 by one tailed t-test compared to vehicle control.