Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol

Abstract

Macrosteatotic livers exhibit elevated intrahepatic triglyceride (TG) content in the form of large lipid droplets (LDs), reduced ATP, and elevated reactive oxygen species (ROS), contributing to their elevated sensitivity to ischemia-reperfusion injury during transplantation. Decreasing macrosteatosis in living donors through dieting has been shown to improve transplantation outcome. Accomplishing the same feat in deceased donor grafts would require ex-vivo exposure to potent defatting agents. Herein, we used a rat hepatocyte culture system exhibiting macrosteatotic LD morphology, elevated TG levels, and elevated sensitivity to hypoxia and reoxygenation (H/R), to test for such agents and ameliorate H/R sensitivity. Macrosteatotic hepatocyte preconditioning for 48h with a defatting cocktail, previously developed to promote TG catabolism, reduced the number of macrosteatotic LDs and intracellular TG levels by 82% and 27%, respectively, but did not ameliorate sensitivity to H/R. L-carnitine supplementation to this cocktail, together with hyperoxic exposure, yielded a similar reduction in macrosteatotic LD numbers, and to a 57% reduction in intrahepatic TG storage, likely by increasing the supply of acetyl-CoA to mitochondria, as indicated by a 70% increase in ketone body secretion. Furthermore, this treatment reduced ROS levels by 32%, increased ATP levels by 27%, nearing ATP levels of lean controls, and completely abolished H/R sensitivity as indicated by ~85% viability post H/R and return of cytosolic lactate dehydrogenase release down to levels seen in lean controls. Cultures maintained for 48h post H/R were ~83% viable and exhibited superior urea secretion and bile canalicular transport compared to untreated macrosteatotic cultures. These findings show that the elevated sensitivity of macrosteatotic hepatocytes to H/R can be overcome by defatting agents, suggesting a possible route for the recovery of discarded macrosteatotic grafts.

Development of a normothermic extracorporeal liver perfusion system toward improving viability and function of human extended criteria donor livers

“…(34) Moreover, normothermic perfusion of steatotic livers from obese Zucker rats using a "defatting cocktail" led to a 50% reduction in intracellular lipid content after 3 hours of machine perfusion. (25) In our preliminary experiments, we employed 2 steatotic human livers not suitable for clinical transplantation and used a defatting protocol at the beginning of NELP. The results demonstrated a 10% reduction in the level of macroglobular steatosis in 1 liver (Table 2).…”

An Update of Liver Transplantation for Nonalcoholic Steatohepatitis

“…The increase in prevalence of hepatic steatosis in the population of the USA in general is reflected in the frequency of fatty liver among potential organ donors. Donor livers with greater than 30 % macrosteatosis exhibit metabolic abnormalities that include increased sensitivity to ischemia, oxidative stress with reperfusion, and increased likelihood of graft complications, including primary non-function [24]. With large populationbased studies observing hepatic steatosis in up to one third of adults in the USA [25], a proportion similar to that reported among living donors, the number of potential donor organs discarded on the basis of fat is likely to increase further.…”

“…With large populationbased studies observing hepatic steatosis in up to one third of adults in the USA [25], a proportion similar to that reported among living donors, the number of potential donor organs discarded on the basis of fat is likely to increase further. The observation that ischemia-reperfusion injury sensitivity can be reversed by reducing stored fat [24,26,27] has led to development of several de-fatting strategies to reduce hepatic steatosis in living and deceased donor livers. For potential living donors with hepatic steatosis, an intense protocol to reduce steatosis in 2-8 weeks, using an exercise program that burns 600 kcal/day, a protein rich diet of 1000 kcal/day, and bezafibrate at 400 mg/day is useful [28].…”

“…Heme oxygenase-1-a microsomal enzyme, bortezomib, a NKkB inhibitor, N-acetylcysteine, a precursor to glutathione, IL-6, and the flavin-antioxidant from tea have been studied alone and in combination. Experimental strategies applied on liver grafts during ex vivo preservation include adding IL-6, epidermal growth factor, insulin-like growth factor or L-carnitine [24,31] to preservation solutions [27]. Venous systemic oxygen persufflation (VSOP) and machine perfusion (MP) preservation (as a means of supplying nutrients, removing waste products, and maintaining hepatic microcirculation) are also under development [26].…”

Control of Ischemia-Reperfusion Injury in Liver Transplantation: Potentials for Increasing the Donor Pool

“…Steatotic livers with their extraordinary sensitivity to IRI seem to improve on hypothermic MP and oxygenated subnormothermic MP in comparison with SCS. First results of treatment of steatotic livers with diverse defatting cocktails while on MP are promising [41,42]. …”