Silencing sirtuins: A novel way to thwart reperfusion injury?
Authors: Pieter Borger, Pierre‐Alain Clavien
Journal: Hepatology (2016)

Although ischemia-reperfusion (IR) injury has been recognized as one of the major causes of complications after liver surgery and studied intensively in many laboratories for almost 3 decades,1 the pathophysiology has been only partially unraveled. Initiated by temporary interruption of the blood supply to the organ, the actual tissue damage results from an excessive activation of innate immunity following reperfusion. Immediately after reoxygenation, a surge of reactive oxygen species activates the Kupffer cells to produce chemokines that function to attract and activate neutrophils.2, 3 Inappropriate activation and homing of neutrophils to the microvasculature of the liver is associated with several pathological manifestations of IR. Neutrophils cause havoc to the liver parenchyma through the release of proinflammatory and tissue-damaging proteins, including proteases and damage-associated molecular patterns. Injured liver cells, in turn, may start to release additional aggravating factors, such as high mobility group box 1 (HMGB1), which, in a self-sustaining process, further exacerbates inflammatory responses in the liver, eventually culminating into cell necrosis and—in the worst case—organ failure. HMGB1 neutralization, as well as adenosine triphosphate supplementation, protects the liver from IR injury and oxygen-associated stress.4 Protection from IR injury may logically be achieved by reducing oxidative processes and/or by silencing the affected metabolic pathways. Caloric restriction ("fasting") with a concomitant reduced oxidative metabolism has been recognized to have liver-protective properties, albeit full understanding of the underlying mechanisms is still elusive. Recently, Rickenbacher et al. reported that fasting for 1 day reduced the level of circulating HMGB1 associated with cytoplasmic HMGB1 translocation and aggregate formation, as well as autophagy. The protective effect of fasting was dependent on the presence of sirtuin 1 (Sirt1), a member of the Sirtuin family of proteins with deacetylase activity. Indeed, Sirt1 inhibition completely abolished the translocation of HMGB1 and abrogated the fasting-induced protection from IR-induced liver injury.5 In this issue of Hepatology, Wang et al.6 provide further evidence that the sirtuins make up a balanced system to regulate cellular and physiological processes associated with oxidative stress and ischemic injury. Their intriguing article shows that sirtuin 2 (Sirt2) aggravates postischemic liver injury by deacetylation of mitogen-activated protein kinase phosphatase (MKP) 1. To appreciate the background concept of this study, it is essential to understand some key regulatory features of the cell. The biological activity of proteins is subject to intricate control mechanisms involving several molecular modifications that shuttle proteins between activated and silent states. Consequently, the activity and function of most proteins highly depends on the modifying molecules they are covalently associated with. Among the best-known examples of protein modification is the attachment of a phosphor group, which is performed by kinase proteins. Usually, the attachment of the phosphor group activates the protein, although examples of the opposite are also known. In addition to phosphorylation, protein acetylation is a common modification to change the activity of proteins. Acetylation (or: ethanoylation) describes a biochemical reaction that substitutes a hydrogen atom for a functional acetyl group provided by acetyl-coenzyme A. Protein acetylation occurs as posttranslational modifications and plays vital roles in the regulation and control of metabolic responses and epigenetic reprogramming. Proteomics studies revealed thousands of acetylated proteins, indicating that acetylation is an essential modification of proteins,7 signifying acetylation as a central mechanism to regulate and control metabolic responses and epigenetic reprogramming of cells. A broad range of substrate-specific acetylating enzymes are active in mammalian cells. A sustainable cellular status demands that protein modifications must be rescindable. To counteract kinase activity, cells trigger phosphatases to remove the phosphorus groups. Likewise, acetylated proteins can be counteracted by specific deacetylation enzymes: sirtuins. Sirtuins (short for silent information regulator factor 2-related enzymes) are a class of nicotinamide adenine dinucleotide-dependent deacetylases, proteins that catalyze the removal of acetyl groups. In mammals, seven sirtuin genes have been identified. The biological roles of Sirt1 have been well studied, and the protein mainly operates in pathways preventing energy depletion and those involved in oxidative stress, apoptosis, and inflammation, in particular, under ischemia/hypoxia conditions.8 Sirt2, which is predominantly located in the cytosol, is the least understood of the seven mammalian sirtuins. Recent findings demonstrated that Sirt2 is involved in mammalian metabolic homeostasis, including adipogenesis, fatty acid oxidation, gluconeogenesis, and in inflammatory and oxidative stress responses.9 Using gain-of-function and loss-of-function studies, Wang et al. now elucidated the role of Sirt2 during hepatic IR injury. They show that overexpression of Sirt2 caused more tissue damage post-IR, whereas reducing Sirt2 levels decreases hepatic IR injury. Furthermore, Sirt2 overexpression enhanced the infiltration of neutrophils in IR-injured liver tissue. Activated neutrophils release several noxious proteins detrimental to liver cells, which can, in addition to tumor necrosis factor alpha, induce apoptotic and necrotic processes in hepatocytes. Indeed, Sirt2 increased apoptosis and cytokine production, suggesting that Sirt2 aggravates hepatic IR injury. Wang et al.'s studies also revealed that the expression pattern of Sirt1 and Sirt2 are distinctly different: During the early phase of hepatic IR injury, Sirt1 is down-regulated, whereas Sirt2 is up-regulated following a pattern that is highly reminiscent of cross-regulation through a negative feedback loop induced by Sirt1. Whether Sirt1 regulates the expression and/or activity of Sirt2—and vice versa—was not the topic of the current study and remains to be elucidated. Still, the article harbors an interesting mechanism that may underlie such cross-regulation. Essentially, Sirt2 was demonstrated to deacetylate MKP1, an essential factor in keeping mitogen-activated protein-kinase (MAPK) activity silenced. The deacetylation of MPK1 through Sirt2 reduces its potential as a MAPK phosphatase, and the removal of phosphor groups from MAPK is hampered. The net effect of Sirt2 activity thus results in the increase of phosphorylation of MAPK and their downstream biological pathways, which, in turn, may modulate the expression of sirtuins.10 Interestingly, Wang et al. demonstrate that Sirt2 overexpression or Sirt2 deletion did not lead to changes in MKP1 messenger RNA (mRNA) and protein levels. Instead, MKP1 was deacetylated in Sirt2-overexpressing liver tissue, whereas its acetylation status was restored in liver tissue of Sirt2 knockouts. Together, these data confirm a posttranslational regulation of MKP1 activity by Sirt2. The study is also of significance, in that it emphasizes the importance of studying protein modifications (acetylation and phosphorylation), rather than only showing mRNA expression patters. Wang et al.'s study informs us about the activated and regulated biological processes that determine protein activity, intracellular changes, and, ultimately, about cell fate and biological consequences (summarized in Fig. 1). Whether Wang et al.'s findings will also be of relevance in human beings remains a matter of debate, unfortunately, because they neither confirmed nor validated the observed protein modifications in liver tissue obtained from humans. Still, in order to comprehend IR-induced liver injury, this type of study is recommended. We would like to encourage investigators to follow the path of Wang et al., extended with studies using human liver tissue, because understanding protein modifications and interaction networks may bring the knowledge to treat IR-induced liver injury. Here, also, this study paves the way in demonstrating that the specific sirt2 inhibitor, AGK2, attenuated aspartate aminotransferase and alanine aminotransferase levels, diminished inflammatory cytokines and chemokines, and reduced the infiltration of neutrophils. In other words, this study further opens the window to the development of agents that specifically block the activity of sirtuins to counteract hepatic injury post-IR. It should be kept in mind, however, that future studies should preferably include at least some human material and experimental evidence for validation purposes. Then, pharmaceutical compounds that specifically inhibit or activate distinct sirtuins, in particular, Sirt1 and Sirt2, may have a much broader application to treat liver diseases than currently envisaged. Pieter Borger Ph.D. Pierre-Alain Clavien M.D., Ph.D. Swiss HPB and Transplant Center Department of Surgery University Hospital Zurich Zürich, Switzerland