Normothermic machine perfusion
Clinical implementation of defatting via normothermic perfusion of steatotic liver grafts, even if successful in laboratory animal experiments, will have to overcome several logistical obstacles. Although the concept of machine perfusion is gaining clinical acceptance, all systems used to date have been with hypothermic rather than normothermic conditions (25,32). The data suggest that hypothermic machine perfusion was beneficial to nonsteatotic livers compared to simple cold storage (25,26). Because the processes of lipid oxidation and export are likely to be significantly slower at lower temperatures, decreasing temperature may not be desirable for defatting, although this question has not been investigated experimentally (29,33). On the other hand, it may be interesting to evaluate supraphysiological temperatures, as it may be possible to trigger heat shock preconditioning, which has been shown to be protective against I/R stress, as discussed previously (21). Nevertheless, once more evidence is available to support the use of hypothermic perfusion; it is likely that clinician investigators will be inclined to move forward into the realm of normothermic perfusion.
Normothermic perfusion systems will need to be further developed into portable perfusion devices that are equipped to handle the rigors of clinical use and transportation by ground and air, a challenging endeavor given their complexity. The reliability of a normothermic perfusion system will require a much higher threshold, since any flow stoppage due to machine failure—either unrecognized or not acted upon—would cause rapidly damaging warm ischemia. In contrast, the same problem in hypothermic perfusion would not be as damaging since such systems have a ‘backup’ static hypothermic mode, in which case, the result would be similar to simple cold storage, which is the current standard used for transplantation (25–27).
Normothermic perfusion may however provide major advantages that could mitigate these concerns. During normothermic perfusion, the liver is in a metabolically active state, which enables thorough evaluation of its function and thus suitability for transplantation well beyond what is possible when livers are stored using simple cold storage or cold perfusion. The benefits of normothermic perfusion as a tool to deliver novel resuscitative interventions will likely result in researchers continuing to push the envelope in developing normothermic techniques applicable to clinical setting (27).
Optimizing the perfusion solution
Ultimately, the goal of liver defatting is to rapidly decrease the proportion of macrosteatotic hepatocytes, while maintaining high viability and functionality. Steatosis is the result of an imbalance between TG synthesis and breakdown processes in hepatocytes (1,2,9,28). Therefore, to achieve significant defatting, the protocol of choice should shift this balance towards more efficient TG breakdown (lipolysis) and excretion of related byproducts, as well as minimizing TG synthesis (9,28,31,34). The pathways responsible for lipid metabolism are well known; however, there is much work to be done on how best to modulate this metabolism using cocktails of agents in order to achieve rapid defatting without adversely affecting viability and other critical liver functions.
There is a considerable body of literature on transcription factors that regulate lipid metabolism in liver; however, transcriptional regulation typically has a response lag time of greater than 6 h. Thus, compounds that target such pathways will be minimally effective over the time scale of ex vivo perfusion. Rather, agents that mediate their effects via posttranslational mechanisms, including signaling effectors, metabolic substrates and cofactors, should be considered for this approach. The major pathways that control TG storage, as well as defatting agents which have been tested to decrease TG storage in a short timeframe are summarized in Figure 2 and briefly discussed here. The control of lipolysis requires cooperation between perilipins, which are proteins associated with the surfaces of lipid droplets where TG is stored, and lipases, which cleave TGs into diacylglycerol, monoacylglycerol, FFAs and glycerol (28,34–36). Indirect activation of protein kinase A by compounds such as forskolin increases phosphorylation of perilipin 5 on the surfaces of lipid droplets and promotes lipolysis (28,36,37). The lipolysis products will readily reform TG unless they are further metabolized and/or excreted from the hepatocytes. This is achieved, in part, by reesterification of lipolysis products into TG to be packaged in VLDL particles, which are then secreted from the hepatocytes (34). The addition of amino acids and choline has been shown to promote the synthesis of apolipoprotein B and phospholipids, respectively, both critical for VLDL assembly and to reduce steatosis (28,38). Another approach to promote defatting is to increase the transport of FFAs to mitochondria where they undergo β-oxidation to generate ATP and CO2, as well as ketone bodies that are excreted from the liver (28,39). A rate-limiting step in β-oxidation is the transport of FFAs from the cytoplasm into the mitochondria, which requires the conjugation of acyl-coA with L-carnitine by carnitine palmitoyltransferase-1(CPT-1) located on the outer mitochondrial membrane (40). In vivo and in vitro studies have shown that supplementation of L-carnitine in the diet or cell culture medium promotes β-oxidation and a reduction in hepatic TG (40).
Figure 2. TG metabolism in steatotic hepatocytes. Lipids are primarily stored in the form of TG inside lipid droplets which are coated with perilipins. Perilipins control access of the stored TG to cytosolic lipases, which liberate free fatty acids (FFA). FFAs can then undergo oxidation through mitochondrial ß-oxidation. FFA and MG translocate to the ER, where they can be reesterified to TG and packaged into VLDL, which is then secreted out of the cells. Major lipid metabolic pathways, including those involved in the removal of TG from steatotic hepatocytes are illustrated. Addition of potential defatting agents that target some of these pathways, such as forskolin, L-carnitine, amino acids and choline, to the perfusate are shown here and discussed further in the text. The dashed arrow represents indirect activation of protein kinase A by forskolin. TG, triglyceride; FFA, Free fatty acid; DG, diacylglycerol; MG, monoacylglycerol; VLDL, very low density lipoprotein; CPT-1, carnitine palmitoyltransferase-1; ER, endoplasmic reticulum; ATP, adenosine triphosphate.
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Targeting multiple routes simultaneously, as illustrated in Figure 2, may provide the most effective approach, but rational design of defatting protocols will require a better understanding of the potential interactions among the relevant metabolic pathways in macrosteatotic hepatocytes. This would be greatly facilitated if a suitable cell culture model of hepatic macrosteatosis were available to perform both screening and mechanistic studies in an efficient manner, prior to testing in actual livers. Although some steatotic hepatocyte culture systems have been described in the literature, they all exhibited microsteatotic, and not macrosteatotic, features (9,28).
Ultimately, surgeons will require proof that defatted livers are indeed similar to normal lean livers before this approach will gain wide acceptance. It will therefore be important to assess the short- and long-term functionality of steatotic livers for which TG content has been dramatically reduced by rapid normothermic ex vivo perfusion defatting in a relatively short period of time. While many challenges need to be overcome, liver defatting is a potentially promising approach to reduce the sensitivity of macrosteatotic livers to I/R injury, and a new modality that may enable the successful recovery of a large number of livers that would otherwise be discarded.